123 publications mentioning rno-mir-29b-1 (showing top 100)

Open access articles that are associated with the species Rattus norvegicus and mention the gene name mir-29b-1. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

Thus, the downregulation of RAX protein that was observed in the rat retina at 28 and 35 days after injection of STZ could be explained by the inhibition of Sp1 expression due to the upregulation of miR-29b.

We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection.

Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ -induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA -dependent protein kinase (PKR) signaling pathway.

Interestingly, the upregulation of miR-29b at 28 and 35 days after the injection of STZ is accompanied by the downregulation of RAX protein.

According to our hypothesis, the upregulation of miR-29b in retinal neurons of diabetic rats may result in down-regulation of RAX which decreases the activated PKR level with subsequent reduction of the activity of the pro-apoptotic PKR signaling pathway.

Based on the finding that the miR-29 family is expressed in the rat retina [14] and that one miRNA has several targets, we hypothesize that miR-29b could regulate the genes in the pro-apoptotic pathways that are involved in the apoptosis of the retinal neurons of STZ -induced diabetic rats.

The analysis of the expression of miR-29b in the retina by qRT–PCR showed that miR-29b is upregulated at 28 and 35 days after STZ injection.

The next step was to examine the potential targets of miR-29b, as it is known that several miRNAs regulate an overlapping set of target genes [12, 13, 28].

These findings suggest that, if the miR-29b target site in the 3′-UTR of RAX mRNA is accessible in vivo, the expression of RAX may be negatively regulated by miR-29b.

Thus, it is possible that in the retina of diabetic rats miR-29b is acting on another target rather than RAX or this PKR activator is an indirect target of miR-29b.

A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis.

Based on the results of this work and that from the literature, it is reasonable to speculate that RAX expression is indirectly regulated by miR-29b in the retinal neurons of the rat (Figure 9).

Considering the localization results from in situ hybridization, it is reasonable to assume that the miR-29b expression detected by qRT–PCR reflects its expression in RGCs and the cells of the INL of the rat retina.

Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT–PCR, reflects their expression in the RGCs and the cells of the INL.

We selected the best predicted targets of miR-29b by comparing the results from the three target prediction databases [23].

The luciferase assay and the overexpression of miR-29b did not validate RAX as a direct target of miR-29b.

The analysis by immunofluorescence demonstrated that RAX protein is expressed in the same specific retinal cell types as that observed for miR-29b expression (Figure 1B and Figure 4B).

The analysis of the expression of miR-29b in the retina by qRT–PCR reflects the expression seen in the RGCs and cells of the INL, as indicated in Figure 1C.

Recently, it was reported that miR-29b indirectly regulates DNA methyltransferase 1 (DNMT1) by targeting the transcription factor Sp1 which binds to the GC boxes in the promoter of the DNMT1 gene in mice and humans [45- 47].

We observed that miR-29b is upregulated (>3 fold) at 28 (p=0.01) and 35 (p=0.05) days after injection of STZ (Figure 2).

We did not confirm the computational prediction that RAX is a direct target of miR-29b.

However, additional experiments are required to confirm our working hypothesis, such as the validation of rat Sp1as a direct target of miR-29b and the evaluation of the expression of Sp1 and phosphorylated (activated) Sp1 in the retinas of normal and diabetic rats.

This suggests that RAX expression is negatively regulated by miR-29b and may represent a mechanism of protection of retinal neurons against apoptosis, which occurs around 35 days after STZ -induced diabetes [41].

Our results revealed that the binding sites for miR-29b are conserved in human and rat, suggesting that miR-29b may also regulate Sp1 expression in rat (data not shown).

Therefore, it is possible that the upregulation of miR-29b at 28 and 35 days may represent part of an adaptive response to protect the retinal neurons against apoptosis by the PKR signaling pathway in diabetic rats.

A: The expression of RAX protein in CJ4 cells transfected with a miR-29b mimic at a concentration of 0, 50, or 75 nM after 24 and 48 h of transfection.

We used three algorithms to computationally predict potential targets of miR-29b.

B: The diagram shows the target sites for the microRNA-29b (miR-29b) paralogs in the 3′- untranslated region (3′-UTR) of PKR associates protein X (RAX) mRNA, including the RNA hybrid-free energy calculations and the theoretical miRNA-mRNA duplex pairing.

The expression profiles of miR-29a and miR-29c were similar to that found for miR-29b (data not shown).

In silico analysis indicated that RAX is a potential target of miR-29b.

A: The firefly luciferase reporter construct (pISORAX plasmid) contains a partial-length 3′-UTR of RAX mRNA in the 5′ to 3′ orientation with respect to the firefly-luc open reading frame with the firefly luciferase start and stop codons and the potential miR-29 target site.

The results of the computational analysis indicated that one of the miR-29 targets is RAX mRNA.

The paired Student t test was performed to ascertain whether there is statistical significance between the miR-29b expression levels in the retinas of diabetic rats and nondiabetic rats.

The firefly luciferase start and stop codons and the potential miR-29 target site are also shown.

The localization of the binding sites of these paralogs of miR-29 to on the RAX 3′-UTR overlap, and there is only a single target site on RAX for each miRNA studied.

The computational prediction programs suggested that RAX is a target of the miR-29 family (miR-29a, miR-29b, and miR-29c), and miR-29b showed the highest score of prediction.

Figure 7PKR associated protein X (RAX) expression in the presence of a microRNA-29b mimic.

Figure 8PKR associated protein X (RAX) mRNA expression in the presence of a microRNA-29b mimic.

The expression analysis of mature miR-29b was performed with quantitative reverse transcription PCR (qRT–PCR) for the retina samples at various intervals after STZ treatment and for the control retinas.

Interestingly, the in silico analysis revealed that one of the potential targets of miR-29b is RAX (PK R -associated protein X), the only known physiologic activator of PKR.

Arrows indicate that miR-29b was expressed in the ganglion cell layer and the inner nuclear layer of the retinas.

The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA -dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats.

Analysis of microRNA-29b expression by quantitative reverse transcription PCR.

Prediction of microRNA-29b targets.

The expression of the mature miR-29b was normalized to β-actin.

We observed, by in situ hybridization, that miR-29b is highly expressed in the neurons of the RGC of diabetic rats.

We found that the three paralogs of miR-29 (miR-29a, miR-29b, and miR-29c) have a complementary sequence to the seed sequence on RAX with minor divergences (Figure 3B), suggesting that the three paralogs potentially target RAX mRNA.

The retinas were dissected and used either for the analysis of RAX mRNA and miR-29 expression and protein analysis or processed for in situ hybridization and immunofluorescence.

Expression of microRNA-29b in retina.

Analysis of protein and mRNA expression levels of RAX, an activator of PKR, in the presence of a microRNA-29b mimic.

The bioinformatic algorithms TargetScan, miRanda, and FindTar were used to predict miR-29 binding sites in the 3′-UTR of RAX mRNA.

To gain more insight into the role played by miR-29b in the retina during the early stage of STZ -induced diabetes, we investigated whether RAX is a direct target of miR-29b.

It should be emphasized that Sp1 is involved in the transcription of several genes regulated by miR-29b [28].

Moreover, our results provide a new focus for future studies and may contribute to the development of new strategies for the treatment of DR, such as the intravitreal injection of miR-29b.

To investigate whether miR-29b directly regulates RAX expression, we used a luciferase reporter assay.

This fragment was amplified by RT–PCR using primers (sense 5′-GCT GAG TGT GGC ATC CAT TT-3′ and antisense 5′-CCA CTT CAC AAA GCT TTG CAC-3′) that produced a 141-bp amplicon spanning nucleotides 189–330 and containing one potential target site for miR-29b.

All reactions were incubated in the Thermo Hybaid PCR Express (Middlesex, UK) at 42 °C for 1 h. After the RT reaction, the cDNA products were diluted to 1:4. In a 10-μl PCR reaction, 4.0 µl of the diluted cDNA was added to 5.0 μl of 2× PCR master mix (TaqMan Universal PCR master mix; Applied Biosystems) and 1.0 μl of the miR-29b primers and TaqMan probe mix; Applied Biosystems).

The reporter plasmid containing the miR-29b complementary sequence was constructed with a fragment of the 3′UTR of RAX mRNA containing the predicted miR-29b binding site.

The miR-29b staining was observed in the cytoplasm, and there was no change in the cellular localization of miR-29b in the retina of normal and diabetic rats.

To perform in situ hybridization of miR-29b, a locked nucleic acid (LNA) -modified, digoxigenin (DIG)-labeled probe was generated by Exiqon (Vedbaek, Denmark).

HEK293 cells were co -transfected with pISO (B) or pISORAX (C) and pRL-TK together with a synthetic miR-29b mimic or dsRNA as a negative control.

After 24 h, when they reached a confluence of 80%–85%, the cells were transfected at two doses (50 nM and 75 nM) with either the synthetic miR-29b mimic or the dsRNA negative control (described above) and 30 µl of Lipofectamine (Invitrogen, Carlsbad, CA) 2000.

The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT–PCR), and the expression of RAX protein was evaluated by western blot.

As a positive control, a U6 probe generated by Exiqon was substituted for the miR-29b probe.

Cellular localization of microRNA-29b in the retina.

These findings prompted us to perform in silico analysis to search binding sites for miR-29b in the 3′-UTR of rat Sp1 mRNA.

The expression of RAX mRNA was evaluated by qRT–PCR in CJ4 cells transfected with an miR-29b mimic or dsRNA (negative control) at a concentration of 50 or 75 nM, 24 h (A) and 48 h (B) after transfection.

After 24 h, the cells were transfected with Lipofectamine 2000 reagent (Invitrogen), 100 ng of the pISO reporter construct (pISO-REPORT-3′-UTR/ RAX or pISO-REPORT empty), 20 ng of the Renilla luciferase control vector, pRL-TK (Promega, Lyon, France); and either a synthetic miR-29b mimic (Dharmacon, Denmark) or a short double-stranded RNA (dsRNA; Dharmacon), as a negative control at two doses (50 nM or 75 nM).

HEK293 cells were co -transfected with the plasmids (pISO-RAX or pISO empty) and a synthetic mimic of miR-29b or dsRNA (negative control).

We used in situ hybridization to determine the cellular localization of miR-29b in the retina of diabetic rats.

The cellular localization of miR-29b is shown in the control rat retinas (Figure 1A) and the diabetic rat retinas on days 6 (Figure 1B), 28 (Figure 1C), and 35 (Figure 1D) post-STZ injection.

The levels of activated Sp1 should be examined to determine if the phosphorylation of pre-existing Sp1 can explain the apparently miR-29b-independent induction of RAX observed at 6 days after injection of STZ.

Recently it was found that miR-29b has a protective effect against the deposition of extracellular matrix in the trabecular meshwork cells when induced by chronic oxidative stress, as observed in glaucoma [15].

We investigated the cellular localization and expression of miR-29b and RAX in the retina of normal and diabetic rats to elucidate their possible involvement in the apoptosis of retinal neurons.

CJ4 cells were transfected with a synthetic mimic of miR-29b or dsRNA (negative control) in concentrations of 0, 50, and 75 nM, and the expression of RAX protein was evaluated at 24 and 48 h after transfection.

We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats.

Figure 1The cellular localization of microRNA-29b in the retina of streptozotocin (STZ) -induced diabetic rats by in situ hybridization.

The specificity of the antibody staining was confirmed by incubating adjacent sections in the absence of the miR-29b antibody.

The distribution of miR-29b was strongest and primarily seen in the ganglion cell layer (GCL).

Abbreviations: Sp1 represents transcription factor Specificity protein 1; miR-29b represents microRNA-29b; RAX represents PKR associated protein X; PKR represents RNA -dependent protein kinase.

The mature miR-29b expression was evaluated by Taqman real-time qRT–PCR in the retinas of normal and diabetic rats.

Recent findings indicated that microRNA-29b (miR-29b) is involved in apoptosis [12, 13].

The indicated concentrations refer to the quantity of miR-29b mimic or dsRNA used for the transfection.

The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively.

The miR-29b signal was weak at 6 days but strong at 28 and 35 days.

2

Conversely, inhibition of mTORC1 signaling resulted in up-regulation of miR-29 expression and suppressed MCL-1 expression in cardiomyocytes.

Moreover, since DM is associated with either insulin deficiency or a lack of insulin signaling, we also posited that insulin would suppress the expression of the miR-29 family in cardiomyocytes and up-regulate MCL-1 expression.

Data presented here shows that insulin down-regulates the expression of diabetic marker miR-29 family miRNAs in mouse cardiomyocytes and preserves the expression of cardioprotective MCL-1. Consistent with this insulin effect, 11-week old hyperinsulinemic ZDF rats only had a mild loss of MCL-1 expression and did not show any damage in myocardium.

Conversely, inhibition of mTORC1 signaling by Rap or inhibitors of mTORC1 substrates significantly increased expression of miR-29 family miRNAs and suppressed cardioprotective MCL-1 in mouse cardiomyocytes.

Since MCL-1 is a target of miR-29 family miRNAs, and Rap treatment increases miR-29 expression, we investigated whether a miR-29 inhibitor cocktail (inhibitors of miR-29a, b and c) would improve MCL-1 expression in Rap treated HL-1 cells.

Since miR-29 suppresses MCL-1 and the miR-29 family miRNAs are elevated in DM we hypothesized that one of the mechanisms by which DM promotes heart disease is by causing dysregulation of the miR-29-MCL-1 axis and suppressing MCL-1 levels in cardiomyocytes that can lead to cardiomyocyte disorganization.

B) Expression of miR-29 family miRNAs (miR-29a, b and c) in mouse cardiomyocyte HL-1 cells is suppressed by treatment with INS (100 nM; 12 h) and up-regulated by treatment with Rap (10 nM; 12 h).

Transfection of HL-1 cells with miR-29 inhibitor cocktail reversed Rap -mediated suppression of MCL-1 expression.

analysis using anti-MCL-1 antibody showed that Rap treatment substantially suppressed MCL-1 expression in HL-1 cells transfected with Allstars negative control siRNA, but not in HL-1 cells transfected with miR-29 inhibitor cocktail (Fig. 2D).

We have shown in vitro that a miR-29 inhibitor cocktail could reverse Rap -mediated suppression of MCL-1 protein expression in cardiomyocytes.

Since increased expression of different members of miR-29 family is associated with DM, we tested the effects of insulin that attenuates the progression of DM, and rapamycin (Rap) that promotes the progression of DM, on the expression of miR-29 family miRNAs in HL-1 cells.

We undertook this study to uncover the role of microRNA miR-29 family and its target MCL-1, a pro-survival molecule that is critical for cardiomyocyte survival under stress, in the myocardium damage seen in diabetic heart disease.

However, suppression of hyperinsulinemia by Rap in the absence of regulation of hyperglycemia as seen in Rap -treated ZDF rat promotes severe dysregulation of cardiac miR-29-MCL-1 axis that leads to disruption and loss of myofibril bundle organization.

This would have led to the up-regulation of all miR-29 family miRNAs in their heart tissues that resulted in the severely dysregulated miR-29-MCL-1 axis in Rap -treated ZDF rats.

Since Rap-treatment increased miR-29 levels and suppressed MCL-1 mRNA levels in mouse HL-1 cardiomyocytes, we tested whether Rap-treatment would increase cardiac miR-29 family miRNAs and suppress cardiac MCL-1 mRNA even further in young ZDF rats.

Further studies are needed to also confirm that in in vivo rodent mo dels a miR-29 inhibitor cocktail would improve cardiac MCL-1 protein expression.

Insulin treatment strongly suppressed miR-29a, b and c in cardiomyocytes whereas Rap treatment significantly enhanced expression levels of all three miR-29 family members in HL-1 cardiomyocytes (Fig. 1B).

20 nM of miR-29 inhibitor cocktail (mirVana miRNA inhibitors for miR-29a, b and c) or 20 nM Allstars negative siRNA (Qiagen) was used for transfection.

Though rapamycin has well-established cardioprotective effects, an additional increase in miR-29 family miRNAs due to mTORC1 inhibition in the heart tissues of DM patients can potentially suppress MCL-1 and exacerbate cardiomyocyte disorganization and cardiac damage.

D) staining with anti-MCL-1 antibody and nuclear stain DAPI in HL-1 cells transfected with either (a) Allstars negative siRNA and (b) Allstars negative siRNA and treated with Rap (10 nM), or (c) miR-29 inhibitor cocktail (mirVana miRNA inhibitors for miR-29a, b and c) and treated with Rap (10 nM).

Since insulin suppressed miR-29 in HL-1 cardiomyocytes, we posited that insulin would improve expression of MCL-1 in these cells.

In brief, the suppression of miR-29 expression by insulin could be a previously unidentified cardioprotective mechanism in hyperglycemia.

Regulation of miR-29 target MCL-1 by insulin and rapamycin in mouse cardiomyocytes.

This observation implied that miR-29 family miRNAs regulate MCL-1expression in HL-1 cardiomyocytes.

Though 11-week old ZDF rats had a mild dysregulation of miR-29-MCL-1 axis that resulted in about 45% suppression of MCL-1, no visible differences were observed in the histopathology of the RV tissues between ZL and ZDF rats.

These data suggested that a miR-29-MCL-1 axis, similar to that seen in mouse and human pancreatic β-cells [15] exists in mouse cardiomyocytes and it is regulated by insulin and rapamycin, an mTORC1 inhibitor.

We only observed a mild dysregulation of miR-29–MCL-1 axis at this stage and a 45% suppression of MCL-1 mRNA (Figs. 3I and 3J).

Therefore, dysregulation of miR-29-MCL-1 axis caused by loss of insulin and mTORC1 inhibition is a major factor in promoting myocardial damage in DM in ZDF rats.

Collectively, our data shows the steps in the dysregulation of miR-29-MCL-1 axis in heart tissues during the progression of DM as shown in Fig. 6. Briefly, at the age of 11 weeks, healthy ZL rats show basal level expression of miR-29 and MCL-1 and their myocardium is well organized.

miR-29 family miRNA expression pattern.

RAP for miR-29 a, b, and c. The role of the miR-29-MCL-1 axis in the progression of DM -associated heart disease is not known.

Since we observed that insulin could suppress miR-29 family miRNAs in cardiomyocyte HL-1 cells, it is conceivable that the lack of significant increase in miR-29c in the myocardium of 11-week old ZDF rats despite severe hyperglycemia could be due to their compensatory hyperinsulinemia (a 14-fold increase in plasma insulin).

Expression of all three miR-29 family miRNAs (a, b and c) were significantly higher (at least 2 fold for each miRNA) in ZDF myocardium.

Though insulin treatment could induce phosphorylation of S6K1 rapidly, in this study we chose a 12 hr treatment to be consistent with the treatment time used for determining the changes in miR-29 and an MCL-1 mRNA expression in response to insulin in HL-1 cells.

Cardiac tissues of 15-week old Rap treated ZDF-rats (Rap treatment from 9-weeks to 15-weeks) displayed a ∼2-fold increase in miR-29 family miRNAs and a 4-fold suppression of MCL-1 mRNA.

Since insulin is an activator of the nutrient sensor kinase mammalian target of rapamycin complex 1 (mTORC1), we further posited that mTORC1-signaling mediates insulin's effects on miR-29-MCL-1 axis.

However, qRT-PCR showed that Rap treated ZDF rats had at least a 2-fold increase in the expression of all miR-29 family members (miR-29a, b and c) (Fig. 4E).

This observation may have important clinical relevance given the fact that patients with DM are reported to have an increase in miR-29 expression [8], [44].

Increased expression of diabetic marker miR-29 family miRNAs is seen in rodent mo dels of DM and in young and adult diabetic patients with T1DM or T2DM.

Therefore, we conclude that regulation of miR-29-MCL-1 axis by insulin is a cardioprotective mechanism and compensatory hyperinsulinemia in conditions of hyperglycemia would regulate miR-29-MCL-1 axis in diabetic heart and prevent significant myocardial damage in young (11- and 15-week) ZDF rats.

Suppression of miR-29 by anti-miR-29 oligomers protects against myocardial ischemia-reperfusion injury, abdominal aortic aneurism and diabetic nephropathy [9]– [13].

HL-1 cells were transfected with either Allstars negative control siRNA or miR-29 inhibitor cocktail and after 8 hours of transfection subjected to treatment with Rap (10 nM) overnight.

Progressive dysregulation of cardiac miR-29-MCL-1 axis in DM and its correlation with cardiac damage.

Based on the data presented here, we contend that the normal functioning of miR-29-MCL-1 axis is an important cardioprotective mechanism regulated by insulin that exists in female mouse atrial cardiomyocytes and male ZDF rat heart tissue.

In contrast Rap -treated ZDF rats have very low INS, severe hyperglycemia, and severe dysregulation of miR-29-MCL-1 axis.

These data suggest that cardiac miR-29-MCL-1 axis is mildly dysregulated in 11-week old ZDF rats that suffer from DM.

Increase in miR-29b leads to the development of aortic aneurisms [10].

I: qRT-PCR analysis data showing the comparative expression levels of miR-29 family miRNAs in myocardium of ZDF rats compared to that in the myocardium of ZL rats.

To our knowledge this is the first report that shows insulin is a regulator of miR-29 family miRNAs.

For this study, we focused on the RV of ZDF rat heart since RV dysfunction from structural and functional perspectives has been described previously in young ZDF rats [5], [6] and therefore the baseline parameters were easy to compare in the context of regulation of the miR-29-MCL-1 axis.

Our in vitro studies on mouse cardiomyocyte HL-1 cells showed that insulin regulates miR-29 family miRNAs (mir-29a, b and c) and improves cardioprotective MCL-1 levels in cardiomyocytes.

Mild dysregulation of cardiac miR-29-MCL-1 axis in a hyperinsulinemic DM background (ZDF rat) does not show significant cardiac myofibril disorganization or loss.

These observations suggest that Rap treatment causes severe dysregulation of the miR-29-MCL-1 axis in cardiac tissues of ZDF rat.

Moreover, Rap treatment of young hyperinsulinemic ZDF rats caused severe dysregulation of cardiac miR-29-MCL-1 axis and myofibril bundle disorganization indicative of myocardial damage.

These observations suggest that the myocardium of Rap -treated ZDF rats that had a further increase in miR-29 a, b and c miRNAs and further suppression of MCL-1 (Fig. 4E and 4F) compared to age-matched control rats, exhibited significant disorganization of myofibril bundles that reflect tissue damage.

Regulation of diabetic marker miR-29 by insulin and rapamycin in mouse cardiomyocytes.

They have mild to moderate dysregulation of miR-29-MCL-1 axis.

These observations revealed that a miR-29-MCL-1 axis exists in cardiomyocytes.

QTLs associated with the rat (rno)-miR-29 a/b cluster located on chromosome 4: 58,107,760-58,107,847 are shown (Taken from Rat RGSC3.4.

Thus, the miR-29-MCL-1 axis is a major contributor to pancreatic dysfunction and T1DM.

In this context, the diabetic marker microRNA miR-29 family that plays a role in increasing cell death is particularly noteworthy.

RAP for miR-29 a, b, and c. The cardiac muscle cell line HL-1 (a generous gift from Dr.

To determine how DM progression (natural or advanced by Rap treatment) caused dysregulation of cardiac miR-29-MCL-1 axis and promoted cardiomyocyte disorganization, we used male ZDF rats, a well-established rodent mo del for advanced DM [5], [6], [26], [27] and evaluated the correlation between regulation of miR-29-MCL-1 axis and disorganization of myofibril bundles in cardiac right ventricle.

miR-29 is also one of the several miRNAs associated with inflammatory microvesicles [14].

In this study we used mouse atrial cardiomyocyte HL-1 cells and right ventricular tissues of ZDF rats to investigate how Rap modulates expression of miR-29-MCL-1 axis.

The miR-29 family consists of miR-29 a, b (b1 and b2) and c that are located on two different chromosomes (chromosomes 4 and 13 in rat, 1 and 6 in mouse and 1and 7 in human) [7].

Further studies with primary cultures of cardiomyocytes from rat atrium and ventricle are needed to confirm that INS -mediated modulation of miR-29-MCL-1 axis is similar in atrial and ventricular cells.

This study was undertaken to investigate whether insulin regulated the miR-29-MCL-1 axis in cardiomyocytes and if conditions that lead to progressive loss of insulin promote dysregulation of cardiac miR-29-MCL-1 axis and disorganization of cardiomyocytes.

3

Our finding that miR-29b-3p was downregulated in vascular calcification was consistent with the results of Du et al. [42], who reported that miR-29b-3p inhibits vascular smooth muscle cell calcification by decreasing the target genes disintegrin and metalloproteinase with thrombospondin motif-7 (ADAMTS-7) expression, in an inorganic phosphorus and chronic kidney disease -induced rat calcification mo del.

The results of this transfection showed that an increase of miR-29b-3p inhibited MMP2 expression and loss of miR-29b-3p -induced MMP2 expression in rat VSMCs, indicating that miR-29b-3p had an effect on MMP2 expression, either directly or indirectly (Figure 4).

Overexpression of miR-29b-3p or inhibition of miR-29b-3p in rat VSMCs resulted in MMP2 downregulation or upregulation at the protein level, respectively.

Subsequent studies reported that miR-29b-3p directly regulated MMP2 expression, as shown by overexpression and inhibition of miR-29b-3p with mimics, inhibitors, and a luciferase reporter assay.

In addition, accumulating evidence suggests that miR-29 downregulation inhibits dilation of aneurysms and is helpful in an early fibrotic response at the aortic wall by increasing target gene expression in murine mo dels of experimental aneurysms and human aneurysm tissues [29].

The transcriptional activity of a MMP2 decrease may have a role for MMP2 downregulation at mRNA and proteins levels in the cholecalciferol -induced rat calcification mo del, but it does not affect the miR-29b-3p inhibition of MMP2 expression at posttranscriptional levels, as shown by transfecting with miR-29b-3p mimics and inhibitors and the luciferase reporter assay.

We therefore speculate that there is an opposite trend among expressions of miR-29b-3p, MMP2, TGF-beta1, and miR-29b-3p downregulation, contributing to vascular calcification by upregulation of MMP2 and TGF-beta1 in the angII -induced calcification mo del.

Recently, mcl, an antiapoptotic Bcl-2 family member, has been also reported to be a target of miR-29b-3p in HeLa and KMCH cells [46], and miR-29b-3p downregulation inhibited apoptosis and promoted cancer cell proliferation.

The relationship between miR-29b-3p and MMP2 was consistent with our finding that miR-29b-3p downregulation resulted in MMP2 upregulation and promoted vascular calcification in rats.

The Effect of Inhibition and Overexpression of miR-29b-3p on MMP2 Expression in Rat VSMCs.

To inhibit miR-29b-3p expression, cells were transfected with 150 nm inhibitors of miR-29b-3p using a similar method as described for the transfection of mimics.

Maegdefessel et al. [29] reported that miR-29b-3p downregulation significantly inhibited abdominal aortic aneurysm (AAA) expansion and progression and promoted fibrosis within AAA walls to decrease the aneurysm rupture rate in two mice AAA mo dels induced with porcine pancreatic elastase and angiotensin II (angII).

Moreover, we investigated whether MMP2 expression correlated with miR-29b-3p levels, which may be involved in regulating target gene expression at the posttranscriptional level.

In recent years, some studies have reported that miR-29 is a novel diagnostic biomarker and therapeutic target for cancer, because it plays an important role in angiogenesis, invasion, and metastasis of tumors by regulating MMP2 expression [30].

Taken together, the results showed that miR-29b-3p was involved in vascular calcification via targeting of MMP2 expression.

Moreover, a luciferase reporter assay was used to show that miR-29b-3p directly inhibited MMP2 expression.

In the present study, we showed that the expression of miR-29b-3p was downregulated in rat mo dels of vascular calcification induced with cholecalciferol when compared with the control group.

Furthermore, there was a negative correlation between miR-29b-3p expression and MMP2 expression.

The difference of miR-29b-3p target genes in the two studies can be explained by the observation that specific miRNAs may target multiple mRNAs and that a specific mRNA may contain binding sites for different miRNAs.

It has been reported that increased miR-29b-3p expression promoted VSMC calcification by decreasing elastin expression in the inorganic phosphorus -induced VSMC calcification mo del [49].

Expression of MP2 and miR-29b-3p were normalized with β-actin and U6, respectively, and the relative expression was determined by the comparative CT (2 [−ΔΔCt]) method.

Similarly, in our study, miR-29b-3p was downregulated during rat calcification compared with the control group, implying that miR-29b-3p inhibited vascular calcification by some unknown mechanism.

3.4. miR-29b-3p Directly Regulates MMP2 Expression.

Notably, upregulation of MMP2 negatively correlated with miR-29b-3p in calcified arterial tissues.

In future studies, we plan to overexpress and knock-out miR-29b-3p with lentivirus in VSMCs and then test whether miR-29b-3p plays a role in potential mechanisms of vascular calcification.

Furthermore, exogenous transfection of miRs was used to determine whether miR-29b-3p can regulate MMP2 expression in rat VSMCs.

These results indicated that MMP2 is a direct target gene of miR-29b-3p.

The relative luciferase activity was significantly inhibited by overexpression of miR-29b-3p in the wild-type 3′-UTR of the MMP2 group compared with the MMP2 3′-UTR -negative control group (P < 0.001) and MMP2 3′-UTR-mutant group (P < 0.001).

Furthermore, all members of the miR-29 family were downregulated in myocardial tissue adjacent to the infarct and during cardiac fibrosis after acute myocardial infarction in mice and humans [28].

The mimic, inhibitor, and control sequences of miR-29b-3p were transfected in VSMCs to change the levels of cellular miR-29b-3p (Figure 3).

Furthermore, miR-29b-3p largely decreased, resulting in myocardial fibrosis of angiotensin II, involved in the TGF- β1/Smad signal pathway in the angII -induced hypertensive heart disease mouse mo del [44].

Mimics (5′-UUGUGACUAAAGUUUACCACGAU-3′) and inhibitors of rno-miR-29b-3p (5′-AACACUGAUUUCAAAUGGUGCUA-3′) and the negative control (5′-UCACAACCUCCUAGAAAGAGUAGA-3′) were transfected into A7R5 VSMCs.

To further characterize the relationship between miR-29b-3p and MMP2, we used bioinformatics and a target prediction database of miRs, to determine that MMP2 was a potential target of miR-29b-3p.

The miR-29 family (miR-29a, miR-29b-3p, and miR-29c) has been previously implicated in multiple pathological changes in cardiovascular diseases.

Our results indicated that miR-29b-3p is involved in the progression of pathological vascular calcification by targeting MMP2.

Correlation analyses showed that there was a negative correlation between MMP2 protein levels and mRNA and miR-29b-3p expression (R [2] = 0.443 and 0.481, resp.

We therefore speculate that miR-29b-3p plays a role in MMP2 expression and is involved in vascular calcification.

For expression, miR-29b-3p and mimics of miR-29b-3p were transfected into cells at a final concentration of 50 nm.

To further characterize the mechanisms involving miR-29b-3p -mediated MMP2 expression, a luciferase assay was performed to determine whether miR-29 can directly target MMP2.

), as shown in Figure 2. These data suggested that miR-29 may be involved in vascular calcification through mediation of MMP2 expression.

The rat miR-29 family includes three members: miR-29a, miR-29b-3p, and miR-29c, whose dysregulation has been reported in many studies.

The results also suggested that miR-29b-3p can negatively regulate vascular calcification.

In addition, the miR-29 family is involved in aneurysm development by mediating extracellular matrix protein synthesis, including Col1a1, Col3a1, Col5a1, and Eln.

However, whether miR-29b-3p can be used as a treatment for vascular calcification both in vivo and in vitro needs to be confirmed with further studies.

2.5. qRT-PCR for MMP2 and rno-miR-29b-3p.

Figure 5 shows that seven bases of rno-miR-29b-3p in the seed region are complementary with MMP2 3′-UTR bases at positions 269–275.

Furthermore, whether miR-29b-3p is a treatment for vascular calcification in vivo and in vitro needs further studies.

To confirm the regulation between miR-29b-3p and MMP2, a luciferase reporter assay was performed in HEK293T cells.

However, in the calcification rat group, miR-29b-3p was significantly decreased (P = 0.0035).

MMP2 and miR-29b-3p are associated with vascular calcification induced by cholecalciferol in rats.

After transfection for 48 h, RNA was extracted using TRIzol® (Invitrogen, Carlsbad, CA, USA), and successful transfection was confirmed by the quantitative real-time polymerase chain reaction (qRT-PCR) for miR-29b-3p.

MMP2 and miR-29b-3p Are Involved in Arterial Calcification.

The relationship between rno-miR-29b-3p and MMP2 mRNA and protein was determined using linear least squares regression analyses.

The cells were grown to 60% confluency in 24-well plates in complete culture medium [high glucose DMEM (Gibco, New York, USA) with 10% FBS, 50  μg/mL streptomycin, and 50 units/mL penicillin] with luciferase reporter plasmids carrying wild-type MMP2 3′-UTR, mutant MMP2 3′-UTR, and a 3′-UTR negative control and rno-miR-29b-3p or rno-miR negative control plasmid and renilla plasmid cotransfected into HEK 293T cells.

4

Since, we did not find upregulation of miR-29b expression in human thyroid samples in which PATZ1 was downregulated (data not shown) it is possible that, similarly to what occurs for other tumour suppressor genes silenced by oncogenic RAS 50 51, the Ras-directed downregulation of PATZ1 proceeds in two steps: PATZ1 may be first downregulated by miR-29b in a Ras -driven reversible step.

Also in these cell clones miR-29b was upregulated and PATZ1 downregulated, showing that miR-29b overexpression and PATZ1 downregulation are persistent events in chronic thyroid cell transformation induced by Ras oncogene.

In breast cancer, miR-29b expression is negatively associated with the HER2 sub-type, and its overexpression inhibits breast cancer cell proliferation and induces apoptosis mainly downregulating STAT3 protein levels 43.

miR-29b and PATZ1 expression levels are inversely correlated in thyroid cells expressing the Ha-Ras [V12] oncogeneInterestingly, miR-29b is one of the most upregulated miRNAs in FRTL5 following expression of a Ras oncogene in an inducible cell system, in which a chimeric form of Ha-Ras oncoprotein, ER-Ras, can be activated by tamoxifen 26, as assessed by screening the www.

In agreement with the ability of miR-29b to target PATZ1, we found that PATZ1 and miR-29b expressions were inversely correlated at both RNA and protein level in a thyroid cell system in which the expression of the oncogenic Ha-Ras [V12] was induced by a tamoxifen-inducible construct, as well as in FRTL5 cells in which Ha-Ras [V12] was stably expressed.

From these data we can assume that miR-29b has oncogenic activity by downregulating the tumour suppressor activity of PATZ1, in agreement with the ability of miR-29b to enhance cell migration and invasion in nasopharyngeal carcinoma progression by regulating SPARC and COL3A1 expression 39.

Moreover, it has also been reported that miR-29b expression is upregulated in rat thyroid cells following cell growth induced by thyreotropin and its overexpression promotes thyroid cell proliferation.

As shown in Fig. 1d, overexpression of miR-29b significantly reduced luciferase activity of the reporter vector, compared to scramble transfected controls, suggesting that the inhibition of PATZ1 protein expression by miR-29b acts, either in a direct or an indirect manner, on the 3′-UTR of PATZ1.

All these signals were drastically reduced (80% reduction) in thyroid cells overexpressing miR-29b, suggesting that all expressed PATZ1 variants may be targets of this miRNA.

Consistently, increased miR-29b expression was found in experimental murine PTU -induced and human goiters, thus indicating its upregulation as a critical event in the regulation of thyroid cell proliferation 33.

Equally, miR-29b expression inhibits glioblastoma cell proliferation, migration, invasion, angiogenesis and stemness maintenance, while promoting apoptosis, by targeting DNMT3A-3B and BCL2L2 44 45.

In Fig. 1a, we show the miR/PATZ1 alignment: the mirSVR downregulation score is −0.1870, predicting PATZ1 as a very likely candidate target of miR-29b.

Among them, we concentrated on miR-29b since it was one of the most upregulated miRNAs in FRTL5 rat thyroid cells following expression of the Ha-Ras [V12] oncogene 28.

Interestingly, miR-29b is one of the most upregulated miRNAs in FRTL5 following expression of a Ras oncogene in an inducible cell system, in which a chimeric form of Ha-Ras oncoprotein, ER-Ras, can be activated by tamoxifen 26, as assessed by screening the www.

To explore a possible interaction between miR-29b and PATZ1 downstream of oncogenic Ras, we analysed the expression of PATZ1 and miR-29b in the same tamoxifen-inducible FRTL5 cell system, in which the expression of the Ha-Ras [V12] oncogene was induced at different time points.

Next, to further assess the inhibition of PATZ1 protein by miR-29b, we used a PGL-3-CTRL vector containing the 3′-UTR sequence common to the hPATZ1-001 and 004 transcripts, which includes a predicted miR-29b target site, cloned downstream the firefly luciferase gene.

The expression of miR-29b and PATZ1 was also analysed in two previously established independent clones of FRTL5 cells stably expressing high levels of the human Ha-Ras [V12] oncogene 26.

Indeed, mir-29b inhibits the progression of esophageal squamous cell carcinoma by targeting MMP-2 42.

miR-29b and PATZ1 expression levels are inversely correlated in thyroid cells expressing the Ha-Ras [V12] oncogene.

Still, miR-29b is expressed at higher levels in indolent human B-cell CLL (chronic lymphocytic leukaemia) with respect to normal CD19 + B cells and, consistently, transgenic mice overexpressing miR-29b in B cells developed B-CLL 41.

Conversely, PATZ1 was down-regulated after treatment with tamoxifen at both mRNA (Fig. 2c) and protein levels (Fig. 2d), confirming the functional miR-29b/PATZ1 interaction in thyroid cells.

Indeed, PATZ1 protein levels were downregulated following thyroid cell transfection with miR-29b synthetic precursor (Fig. 2a).

Interestingly, differently from HEK293 and PC Cl3 cells, transfection of miR-29b precursor into FRTL5 cells, another rat thyroid cell line, resulted in downregulation of PATZ1 at mRNA level (Fig. 2b).

As shown in Fig. 2, consistent with the data extracted from the above website, miR-29b was upregulated following induction of Ha-Ras [V12] as early as after 24 h of treatment with tamoxifen (Fig. 2c).

Here, we focused on miR-29b since it has been previously found up-regulated in thyroid cell proliferation 33 and transformation 28.

To rule out the possibility that such effects could be due to tamoxifen treatment, we treated both ER-Ras FRTL5 and parental FRTL5 cells with tamoxifen, showing that miR-29b is induced and PATZ1 downregulated only in the presence of oncogenic Ras activity (Fig. 2d).

Conversely, PATZ1 transcript levels were significantly downregulated only in FRTL5 cells, suggesting that miR-29b ability to reduce PATZ1 mRNA stability is cell context -dependent.

As these cells are transformed by the native human Ha-Ras oncogene, it is ruled out the possibility that induction of miR-29b, as well as downregulation of PATZ1 could be an artefact of the chimeric Ras oncoprotein.

Both these variants were downregulated after transfection of the miR-29b (see Supplementary Fig. S3).

miR-29b targeting of PATZ1 in rat thyroid cells.

However, it is worth to note that miR-29b acts as a tumour suppressor in other cancer tissues.

Similar tumour suppressor activity for miR-29b in colon 46, lung 47 and ovarian 48 cancer tissues was reported.

Here, we show that enforced miR-29b expression in thyroid and non-thyroid cells significantly reduces PATZ1 protein levels.

miR-29b and PATZ1 expression levels were normalized for endogenous U6 and G6PD levels, respectively.

All together these results confirm that miR-29b targets PATZ1 in thyroid cells and suggest that PATZ1 is a downstream effector of the oncogenic Ras signalling.

Taking together the qRT-PCR results from both the inducible and stable clones we found a strong negative correlation (r = −0.797), meaning a functional dependence between miR-29b and PATZ1 expression.

These data are consistent with data extracted from the DIANA-TarBase v7.0 database 34, where PATZ1 is indicated as an experimental validated target of has-miR-29b-3p as assessed by immunoprecipitation in BCBL-1 lymphoma cells 35.

Notably, it was the miR-29 isoform more expressed in this Ras -driven rat thyroid cell transformation system.

Moreover, miR-29b enhances migration of human breast cancer cells and is overexpressed in breast metastases in comparison with the breast cancer tissue 40.

PATZ1 is a target of miR-29b.

The ability of miR-29b to target PATZ1 was also confirmed in a thyroid cell system using the rat thyroid cell line PC Cl3.

Validation of PATZ1 as a target of miR-29b.

It is likely that the cellular context determines the oncogenic or the antioncogenic activity of miR-29b, as previously reported for other miRNAs 49.

Interestingly, the mRNA levels of PATZ1 in Ha-Ras [V12] stable clones show a perfect negative correlation (r = −1) with the levels of miR-29b (Fig. 2c).

This reporter vector was transfected in HEK293 cells along with synthetic precursor of miR-29b, or scramble miRNA control, and luciferase activity was assessed 72 h after the transfection.

PATZ1 protein levels were reduced up to the 60% in cells transfected with miR-29b in comparison with the cells transfected with the scrambled oligonucleotide.

The mean ± SE of one experiment performed in triplicate and three independent experiment performed in duplicate is reported for miR-29b and PATZ1, respectively.

However, Independently from the role of miR-29b, the results reported here suggest that PATZ1 may be a negative effector of the oncogenic Ras signalling in thyroid carcinogenesis.

HEK293 transfections were performed by Lipofectamine 2000 (Invitrogen) according to manufacturer’s instruction, with 100 nM Scramble or 100 nM miR-29b miRNA precursors (Ambion, Austin, TX) together with PGL-3-CTRL vector or the PATZ1-3′UTR luciferase reporter plasmid.

5

Taken together, the present study shows that bladder outlet obstruction, such as that seen in elderly men with enlarged prostate glands, leads to reduced expression of miR-29b and miR-29c in the bladder and that this is associated with increased expression of miR-29 targets, including the matrix proteins elastin and Sparc.

Human detrusor cells were transfected with miR-29c inhibitor and eight validated miR-29 targets, most of which were represented among the top 50 predicted targets in Figure 3A, were examined using western blotting.

We also demonstrate that genetic depletion of miRNAs, including miR-29, increases bladder elastin expression and stiffness independently of outlet obstruction and that miR-29 inhibitor transfection in vitro replicates several of the expression changes associated with miR-29 repression in outlet obstruction.

Together, these circumstances may well explain the more widespread apparent impact of miR-29 repression in outlet obstruction (8/8 examined target proteins increased) compared to inhibitor transfection (4/8 examined target proteins increased).

Four of the eight selected miR-29 targets, including Eln (elastin or tropoelastin), Fos (also known as c-Fos), Sparc (osteonectin) and sprouty homolog 1 (Spry1), were significantly increased following inhibitor transfection (Figure 3D, left row).

To address the functional impact of miR-29, we transfected a miR-29 inhibitor and mimic in vitro and conditionally deleted Dicer in vivo [5] and examined the effect of these interventions on tropoelastin expression and on tissue mechanical properties.

Currently, we can only speculate on the role of these proteins in outlet obstruction, but it is of considerable interest that the miR-29 target Spry1 [14], which is an established ERK1/2 inhibitor [49], increases after prolonged outlet obstruction.

For tropoelastin, whose mRNA correlated inversely and significantly with miR-29 in outlet obstruction, we found that transfection of a miR-29c inhibitor resulted in increased tropoelastin expression.

Of the 30 miRNAs that are highly expressed in the mouse detrusor [5, 41] none except miR-29 is predicted to target tropoelastin.

Some predicted and confirmed miR-29 targets, including Fbn1, which is an integral part of the elastic fiber meshwork, and Lamc1, a protein present in the basement membrane, were increased in outlet obstruction but were not significantly affected at the protein level following inhibitor transfection (not shown).

To address whether reduced miR-29b/c following outlet obstruction was associated with altered expression of target mRNAs we did an mRNA microarray experiment.

Additional regulatory inputs on miR-29 expression include c-Myc and NF-κB [11], and recent work has provided considerable insight into c-Myc -mediated repression, which appears to depend on a repressor complex consisting of c-Myc, histone deacetylae 3 (Hdac3) and enhancer of zeste homologue 2 (Ezh2) [18].

Detrusors from smooth muscle-specific Dicer knockout mice were used to examine if reduction of miR-29 in vivo increases tropoelastin expression.

We found that Dicer knockout reduced miR-29b and miR-29c by about half and increased tropoelastin expression.

The extracellular matrix molecule elastin is one of the best established targets of miR-29, and its message has 14 binding sites dispersed over the coding sequence and the 3’UTR [12].

The initial association found for miR-29b/c is illustrated in Figure 3A which shows that miR-29b/c targets were elevated at 10 days (vs.

MiR-29b and miR-29c were among the 63 differentially expressed miRNAs in outlet obstruction (q=0; n=6−8; GEO accession number GSE47080).

MiR-1 (not shown), miR-29b, and miR-29c returned significant associations with target mRNA levels.

Time courses of expression for miR-29c and miR-29b from the microarray experiment are depicted in Figure 2A and 2B.

Real-time quantitative PCR for miR-29c and miR-29b (Figure 2C and D) confirmed reduced expression of both miRNAs at 10 days.

Repression of miR-29 after outlet obstruction is associated with increased levels of miR-29 target proteins.

Outlet obstruction and transforming growth factor β (TGF-β1) stimulation leads to reduced expression of miR-29.

0082308.g005 Figure 5(A) Western blots for eight miR-29 targets in sham-operated control bladders and at 6 weeks of obstruction.

We also examined if the reduction of miR-29 correlated with altered miR-29 target mRNAs, including tropoelastin and Sparc.

6 weeks) of the top 50 mRNA targets of miR-29 when miR-29 was repressed at 10 days and when miR-29 recovered after de-obstruction (c. f. Figure 2A and B).

Stimulation with TGF-β1 for 48h led to reduced expression of miR-29c and miR-29b (Figure 2E and F).

The experimental support for an impact of miR-29 on protein synthesis in the bladder following outlet obstruction extends well beyond a significant correlation between miR-29b/c and target mRNAs.

Expression of (E) miR-29c and (F) miR-29b in vehicle -treated (control) and TGF-β1 -treated human urinary bladder smooth muscle cells.

sham) of miR-29 target messenger RNAs (mRNA; black circles) and proteins (white circles).

Considerably more time was moreover allowed for de-repression of miR-29 targets in the in vivo setting (6 wk vs.

We first examined expression of miR-29b and miR-29c following 5 weeks of Dicer deletion and found both miRNAs to be reduced (Figure 6A, B).

It may be argued that a miRNA other than miR-29 is responsible for altered tropoelastin expression in Dicer KO bladders.

Outlet obstruction and TGF-β reduce miR-29 expression.

0082308.g002 Figure 2Time courses of (A) miR-29c and (B) miR-29b expression following rat bladder outlet obstruction.

This revealed a pronounced increase around individual cells and around muscle bundles in obstructed bladders, consistent with its increased mRNA level and with the fact that it is a miR-29 target [48].

We next set out to determine miR-29 expression.

Real-time quantitative PCR to confirm reduced expression of miR-29.

We next tested whether TGF-β1 reduces miR-29 expression using cultured smooth muscle cells from human bladder.

Repression of miR-29 during outlet obstruction is associated with increased levels of miR-29 target messenger RNAs (mRNAs).

The matricellular protein Sparc is a confirmed miR-29 target [13] that influences collagen fibril morphology and function [47].

Bladder outlet obstruction increases miR-29 target protein levels.

Type I and type III collagens are however established targets of miR-29 [10], and their mRNAs were largely unchanged at 10 days and at 6 weeks (Col1a1 at 10 days: up by 15%, q=2.7, p=0.05; Col3a1 at 10 days: up by 12%, q=8.7, p=0.12).

Unlike other miRNAs, miR-29 also targets a large battery of collagens, including collagens I and III [10].

This would repress miR-29, and hence it would be more difficult to see an effect of miR-29 inhibition.

Our studies support a mo del in which multiple signaling pathways converge on repression of miR-29 in outlet obstruction, facilitating matrix protein expression and leading to altered mechanical properties of the urinary bladder.

Time courses of (A) miR-29c and (B) miR-29b expression following rat bladder outlet obstruction.

MiR-29 target mRNAs change in outlet obstruction.

Thus, several independent lines of evidence support a regulatory role of miR-29 in tropoelastin synthesis in the bladder, consistent with the large number of miR-29 binding sites in its mRNA [10, 12].

Several of the miR-29 targets that we studied, including Col15a1, Tdg and Spry1, have not been considered previously in the context of hypertrophic growth and remo deling of the bladder.

Taken together these findings strongly support the view that the aforementioned repressor complex, assembled upon accumulation of c-Myc/Ezh2 in outlet obstruction, regulates miR-29b in the detrusor.

SMAD proteins belong to a conserved family of TGF-β signal transducers that are regulated by phosphorylation [17], and the repression of miR-29 by TGF-β was shown to involve SMAD3 [16].

The matricellular protein Sparc has three miR-29 binding sites clustered in its proximal 3’ UTR, and, similar to elastin, this protein is effectively regulated by miR-29 in vitro [13].

Real-time quantitative polymerase chain reaction (n=5-7) for miR-29b (A), miR-29c (B) and elastin (Eln, C) in control (Ctrl) and Dicer knockout (KO) mouse bladders.

A correlation between the c-Myc mRNA and miR-29b was moreover seen, and we directly demonstrate accumulation of c-Myc and Ezh2 using western blotting.

Our starting hypothesis was that TGF-β/SMAD3 signaling would repress miR-29 in outlet obstruction.

Figure S2 Flow chart showing the mo del proposed for miR-29 repression in outlet obstruction and for miR-29 -mediated matrix remo deling and altered passive mechanical properties.

The reduced level of miR-29 leads to increased levels of mRNAs encoding extracellular matrix proteins (3), including elastin and Sparc (osteonectin), but possibly also collagens and fibrillin-1. The resulting protein synthesis and matrix deposition (4) leads to increased detrusor stiffness (5) (and increased elastic modulus) which counteracts (6) further distension.

Together, these findings support the view that repression of miR-29, independent of surgical obstruction of the urethra, leads to matrix remo deling.

We therefore hypothesized that outlet obstruction leads to SMAD3 phosphorylation repressing miR-29, and that this in turn has an impact on protein synthesis and mechanical properties of the bladder.

Studies using cultured cells support the idea that transforming growth factor-β (TGF-β), a central mediator in fibrogenesis, represses miR-29 [16].

Therefore, reduction of miR-29b and miR-29c could well promote collagen production at an unchanged mRNA level at these times.

sham), a time point when miR-29 was reduced (c. f. Figure 2A and B).

The miR-29 cluster has gained recognition as a modulator of extracellular matrix production [7- 10].

This hypothesis was based on a handful of prior studies demonstrating increased mRNA levels for different TGF-β isoforms shortly after outlet obstruction (e. g. 24), and on the documented repression of miR-29 by TGF-β/SMAD3 [11].

Eln correlated significantly with miR-29c (Figure 3E) and with the mean of miR-29b and miR-29c (not shown).

Thus we measured the eight validated miR-29 target proteins (the same ones measured after inhibitor transfection) at 6 weeks of obstruction.

When miR-29b/c increased on de-obstruction (vs.

c-Myc and NF-κB are also known to repress miR-29 [11, 18], and both pathways have previously been shown to be activated in outlet obstruction and by mechanical distension [37- 39].

The proposed mo del fits the data presented in this article, but alternative interpretations are possible and steps upstream of miR-29 repression need in vivo corroboration.

SMAD3 activation, which is known to be involved in TGF-β -mediated repression of miR-29, was not significantly increased at 10 days when miR-29b and miR-29c appeared to be maximally repressed.

The Myc mRNA declined below the control level on de-obstruction, resulting in a significant and inverse correlation with miR-29b (Figure 4B).

c-Myc -mediated repression of miR-29 involves a complex consisting of c-Myc (Myc), histone deacetylase 3 (Hdac3) and enhancer of zeste homolog 2 (Ezh2) which binds to conserved sequences in the promoters of the miR-29a/b1 and miR-29b2/c genes [18].

MiR-29 -mediated extracellular matrix remo deling has been demonstrated in the infarcted heart [10] and during aortic aneurysm progression [7- 9], but miR-29 also plays roles in cell proliferation, muscle differentiation and apoptosis [11].

This in turn (2) activates multiple signaling pathways including c-Myc, NF-κB and TGF-β/SMAD3 that in turn repress miR-29.

We therefore propose that c-Myc/Hdac3/Ezh2 and NF-κB are jointly responsible for repression of miR-29b and miR-29c at 10 days and that SMAD3 is responsible for the sustained repression of miR-29c.

Combined, these findings provide support for our hypothesis that miR-29 reduction contributes to increased protein synthesis in the bladder following outlet obstruction and that this in turn influences matrix properties and stiffness (Figure S2).

We hypothesized that miR-29 repression may contribute to increased detrusor stiffness in outlet obstruction.

We propose that bladder distension leads to repression of miR-29 via three distinct mechanisms and that this has an impact on tropoelastin and Sparc synthesis and on tissue mechanical properties.

To address this hypothesis we examined if SMAD proteins are phosphorylated and whether miR-29 is reduced in outlet obstruction.

Sparc correlated with the mean of miR-29b and miR-29c (Figure 3F).

De-repression of Sparc may thus also contribute to a miR-29 -mediated change of detrusor stiffness in outlet obstruction.

It comprises three miRNAs (miR-29a, miR-29b, and miR-29c) derived from two independent genes [10].

Our lack of evidence for SMAD2/3 activation at 10 days, when miR-29b and miR-29c appeared maximally repressed, forced us to consider alternative mechanisms.

In view of this outcome we tested if Eln and Sparc mRNAs were individually correlated with miR-29 in outlet obstruction.

Added to the correlation between miR-29c and Col4a1 mRNA this favors a causal relationship between the repression of miR-29 and the increase of Col4a1 in outlet obstruction.

Independent confirmation of reduced (C) miR-29c and (D) miR-29b in the obstructed bladder by real-time quantitative polymerase chain reaction (n=6).

This supported the possibility that c-Myc and NF-κB, but not SMAD3, might be involved in the repression of miR-29b and miR-29c at 10 days.

The impact of miR-29 in outlet obstruction is likely underestimated by measuring levels of mRNAs because an important mechanism of miRNAs is translational repression.

6

Recently, altered expression of miRNAs has been shown to mediate myocardial fibrosis post-MI [3], [8], which suggest that use of miR-29b, as a fibroblast-enriched miRNA [11], is a potential anti-fibrosis target therapy based on its ability to directly target mRNAs that encode ECM proteins involved in fibrosis.

Furthermore, other ECM proteins including Elastin and fibrillin 1, as the predicted targets of miR-29b, were significantly down-regulated in miR-29b overexpressing group (data not shown).

We discovered that overexpression of miR-29b significantly down-regulated collagen synthesis during anoxia through inhibited pERK activity as well as MRTF-A, a serum response factor (SRF) serving as a cofactor that responds to TGF-β in fibroblasts, contributing to SMA-enriched myofibroblasts [22].

Recently, van Rooij, et al. [8] reported that miR-29b targets and inhibits a group of mRNAs that encode cardiac fibroblast proteins involved in fibrosis, and that the down-regulation of miR-29b after MI correlated with increased collagen types I and III, and fibrillin 1 in the peri-infarct and remote normal heart regions.

Importantly, the effect of miR-29b overexpression on the fibrotic related genes MRTF-A, collagen I, and collagen III was abolished in CFb in presence of the selective inhibitor of pERK, PD98059 (Fig. 1D1–3).

Among myocardial infarction-regulated miRNA members, the miR-29 family (miR-29a, miR-29b copy 1 and copy 2, and miR-29c) is down-regulated in the peri-infarct region of the heart [8], which is associated with collagen production by fibroblasts, subsequent collagen deposition, and eventually leads to heart failure [11].

In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium.

There are three major findings of this study: 1) Overexpression of miR-29b, by blocking the activation of the p42/44 MAPK-MRTF signaling pathway, inhibits myofibroblast formation and attenuates collagen synthesis.

Since miRNAs target not only single genes but also functionally related gene networks, the paracrine effect of miR-29b overexpressing fibroblasts may contribute to increased iPSC [NCX1+] migration and survival.

miR-29 overexpression in combination with the Tri-P may facilitate iPSC [NCX1+] migration and survival, as well as permit neovascularization of the infarct, leading to improvements in LV functional performance after the occurrence of ischemic heart disease.

This study demonstrated that miR-29b overexpression is an emerging supplement to iPSC -based cell therapy for ischemic heart disease.

Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remo deling.

4 weeks post-MI, slight down-regulation of miR-29b was detected in the infarct areas, but was not statistically different from the Sham group (Fig. 2A).

Rats were assigned to experimental groups, as follows: 1) Sham operated rats had a loose suture placed around the left anterior descending (LAD) coronary artery (Sham group), 2) negative mimic pretreated rats with MI operation followed by Tri-P graft (Ctrl+MI+Tri-P), 3) miR-29b overexpression pretreated rats with MI operation followed by Tri-P graft (miR-29b+MI+Tri-P), 4) miR-29b knockdown pretreated rats with MI operation followed by Tri-P graft (Anti-29b+MI+Tri-P).

These suppressive effects were reversed in miR-29b knockdown cells (CFb [Anti-29b]), which exhibited significant increases in pERK, MRTF-A, collagen I, and III levels (p<0.05).

Taken together, these findings suggest that miR-29b and its downstream target genes, as supplements to cell -based therapies, serve as a promising therapeutic regulator to retard, limit or reverse fibrosis post-MI.

To further explore the in vivo findings, several regions of the infarcted rat hearts including the infarcted region (IF), border region (B) and remote region (R) were harvested to analyze the effect of miR-29b overexpression or knockdown on cardiac fibrosis.

Sham, sham operated group with loose suture around LAD; Ctrl, intramyocardial gene delivery with control; miR-29b, intramyocardial gene delivery with miR-29b-1 mimic; Anti-29b, intramyocardial gene delivery with miR-29b-1inhibitor.

The negative mimic, rno-miR-29b mimic (Dharmacon) or rno-miR-29b inhibitor (Exiqon) were added at the required final concentration (200 nmol/L for each well) after mixing with DharmaFECT Duo Transfection Reagent according to the manufacturer's instructions.

A tiny 32 gauge catheter containing 50 µl of concentrated negative mimic, miR-29b mimic, or inhibitor (25 µM) was advanced from the apex of the left ventricle to the aortic root.

Sham, sham operated group with loose suture around LAD; Ctrl, intramyocardial gene delivery with control mimic; miR-29b, intramyocardial gene delivery with miR-29b-1 mimic; Anti-29b, intramyocardial gene delivery with miR-29b-1inhibitor.

2) Reduced collagen deposition associated with miR-29b overexpression in scar tissue after MI facilitates iPSC [NCX1+] penetration from the Tri-P into the infarcted area, and results in restoration of LV function after MI.

The aim of this study was to determine if miR-29b overexpression in the rat heart in vivo would effectively reduce barrier formation (collagen deposition) after MI and thereby enhance the efficacy of the iPSC-derived Tri-P based cell therapy in improving heart function after regional MI.

In vivo experimental design: negative mimic (Ctrl), miR-29b mimic (miR-29b), and miR-29b inhibitor (Anti-29b) were delivered into the rat hearts before MI as described previously [18].

To probe the miR-29b-triggered downstream signal pathway involved in cardiac fibrosis in vitro, we isolated cardiac fibroblasts and transfected them with negative mimic (Ctrl), or miR-29b mimic (miR-29b), or miR-29b inhibitor (Anti-29b).

0070023.g001 Figure 1Effect of miR-29b overexpression in vitro on cardiac fibrosis.

Effect of miR-29b overexpression in vitro on cardiac fibrosis.

Thus, we speculated that miR-29b overexpression might reduce heart tissue collagen and thereby lower the barrier to progenitor cell engraftment and survival.

This favorable anti-fibrotic effect of miR-29b on fibrosis was reversed by the pERK inhibitor, PD98059, suggesting the crucial role of ERK signaling pathway in fibroblast proliferation, and myofibroblast differentiation [2], [23], [24].

Sham, sham operated group with loose suture around LAD; Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR-29b mimic pretreaed rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft.

In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI).

Compared with sham operated hearts (Sham group), a significant down-regulation of miR-29b was observed in the hearts 3 days post-MI (p<0.05) (Fig. 2A), which was most prominent in the infarcted area (Fig. 2B).

In combination with the Tri-P, overexpression of miR-29b was accompanied by enhanced angiomyogenesis in the underlying infarct region and functional restoration of the LV after MI.

To further study the molecular signaling pathway involved in fibrosis, CFb were transfected with negative mimic (CFb [Ctrl]), miR-29b mimic (CFb [miR-29b]), or miR-29b inhibitor (CFb [Anti-29b]).

To elucidate how miR-29b modulated molecular mechanisms involved in cardiac fibrosis modulated by assigning cardiac fibroblasts (CFb), the subjects were divided into the following treatment groups, 1) negative mimic (CFb [Ctrl]) served as control, 2) mimic microRNA-29b-1 (CFb [miR-29b]), or 3) miRCURY LNA™ microRNA-29b-1 inhibitor (CFb [Anti-29b]).

Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSC [NCX1+] penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.

To further analyze whether miR-29b overexpression interfered with post-MI collagen deposition in vivo, intramyocardial gene delivery was performed (Fig. 2C-1).

However, no obvious anti-apoptosis effect of miR-29b overexpression was observed in comparison to Ctrl group and Anti-29b group.

Effect of miR-29b in vivo on cardiac fibrotic gene related expression.

0070023.g002 Figure 2Effect of miR-29b in vivo on cardiac fibrotic gene related expression.

MiR-29b has been reported to directly target mRNAs that encode various ECM proteins involved in fibrosis [3].

3) Overexpression of miR-29b enhances new vessel formation from the cell patch and these vessels connect to the native coronary circulation.

However, under anoxia CFb overexpressing miR-29b (CFb [miR-29b]) exhibited a significant decrease (p<0.05) in pERK (Fig. 1C1–2), myocardin-related transcription factor-A (MRTF-A, Fig. 1D-1), collagen type I (Fig. 1D-2), and collagen type III (Fig. 1D-3).

To analyze the role of miR-29b on cardiac fibrosis, the total mRNA of rat neonatal cardiomyocytes (neoCM) and CFb were isolated respectively, and qPCR revealed that miR-29b was expressed significantly more in CFb than in neoCM (p<0.05, Fig. 1A).

Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR-29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft.

Ctrl+MI+Tri-P, control pretreated rat with Tri-cell patch graft; miR-29b+MI+Tri-P, miR-29b mimic pretreated rat with Tri-cell patch graft; Anti-29b+MI+Tri-P, miR-29b inhibitor pretreated rat with Tri-cell patch graft.

Quantitative data for fibrosis related genes including exprression of TGF-β1 (C-2), SDF-1α (C-3), MRTF-A (C-4) collagen I (C-5), and collagen III (C-6) in infarcted heart 3 days after intramyocardial gene delivery of control mimic (Ctrl), miR-29b1 mimic (miR-29b), or miR-29b1 inhibitor (Anti-29b).

MiR-29b overexpression reduces the barrier (collagen deposition) to iPSC engraftment from epicardial apposed myocardial tissue progenitor cell patches.

Furthermore, an increased number of newly formed blood vessels, identified by GFP expression, were detected in miR-29b pretreated hearts compared with the other two groups 4 weeks after Tri-P application (Fig. 4E and 4F-2).

Quantitative RT-PCR showed that the expression of miR-29b was significantly increased in CFb [miR-29b] group (p<0.01), and was significantly reduced in CFb [Anti-29b] group as compared with CFb [Ctrl] group (p<0.05, Fig. 1B).

The number of green fluorescent protein positive (GFP [+]) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups.

miR-29b -mediated fibrotic size facilitates mobilization of iPSC [NCX1+] and angiomyogenesis.

Effect of miR-29b on fibrotic size, iPSC migration, and angiogenesis in the infarcted heart 4 weeks after Tri-P graft.

With regard to the function in cardiac fibroblasts, miR-29b may attenuate scar barriers to progenitor cell infiltration thereby facilitating iPSC [NCX1+] penetration from the Tri-P into the infarcted area.

To analyze the effect of anti-fibrosis treatment on heart function, additional infarcted hearts treated with negative mimic, or miR-29b mimic or miR-29b inhibitor were evaluated by echocardiography.

Effect of miR-29b in vivo on cardiomyocyte apoptosis.

However, a complex vascular network was detected in the cell patch at 4 weeks after patch transplantation, which was collateral with native coronary arteries (Fig. 5A) and exhibited greater vessel volume and vessel number (Fig. 5C-1), the hallmarks of the dynamic angiogenic process that was much more robust in the miR-29b pretreated group than in other groups (Fig. 5C-2).

Fluorescence microscopy demonstrated newly formed vessels confirmed by GFP antibody (green color) in the cell patch of miR-29b+MI+Tri-P group (A-2) and Anti-29b+MI+Tri-P group (B-2).

CFb [miR-29b].

The results of the present study demonstrate a distinctive role for miR-29b -mediated collagen deposition in the animal mo del of acute MI followed by iPSC derived Tri-P for the repair of myocardial infarction.

0070023.g003 Figure 3Effect of miR-29b in vivo on cardiomyocyte apoptosis.

The anti-fibrosis effect of miR-29b pretreatment was further supported by picrosirius red staining under polarized light, which showed significantly lower levels of cardiac fibrosis 4 weeks after Tri-P graft (Fig. 4A-1).

However, no obvious heart functional changes were observed in miR-29b alone treatment group (data not shown).

miR-29b group.

The pretreatment strategy of intramyocardial delivery with miR-29b followed by Tri-P, in the setting of MI, resulted in restoration of LV mechanical function after MI.

Although Tri-P transplantation induced a robust neo-vascularization as early as 1 week post-patch graft, no differences in vessel volume or vessel number were detected between the miR-29b pretreated group and the Anti-29b pretreated group at that early time point (data not shown).

Micro-CT imaging for collateral circulation formation in miR-29b+MI+Tri-P (A) and Anti-29b+MI+Tri-P group (B).

LV fibrosis, analyzed by Masson's Trichrome staining 4 weeks after patch transplantation, was significantly reduced in the miR-29b pretreated group.

In the current study, we observed a significantly increased number of GFP [+] myocytes (identified by cTnT staining) in the infarcted region of the miR-29b pretreated group.

miR-29b -mediated fibrotic size facilitates mobilization of iPSC [NCX1+] and angiomyogenesisRats were subjected to LAD ligation for 7 days, followed by apposition of the Tri-P to the infarcted heart region.

0070023.g005 Figure 5Micro-CT imaging for collateral circulation formation in miR-29b+MI+Tri-P (A) and Anti-29b+MI+Tri-P group (B).

CFb [miR-29b]; [‡]p<0.05 vs.

Our in vivo findings demonstrate that the post-MI fibrotic processes can be reversed by miR-29b intramyocardial delivery leading to decreased synthesis of MRTF-A, collagen I and III in the infarcted/border regions.

Collagen synthesis is mediated by miR-29b in the heart fibroblasts.

miR-29b+MI+Tri-P group.

By micro-CT analysis, we demonstrated collateral blood vessel network formation arose from the cell patch and connected to native coronary arteries as a prominent feature in miR-29b pretreated hearts in contrast to the Anti-29b group.

Furthermore, GFP positive labeling (Fig. 5C-3) confirmed significant vascularization in the Tri-P region in the miR-29b pretreated group.

7

Conversely, upregulation of miR-29 levels was observed after stimulation of HSC with the antifibrotic mediator HGF (Figure 8), previously shown to inhibit expression of various collagens [21], [48], [49].

Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

Indeed, our in vitro data reveal a definite inhibition of collagen type IV, that is the most upregulated collagen form in the fibrotic liver [6], by miR-29.

Whereas TGF-β stimulation leads to decreased miR-29 levels, but to pronounced upregulation of collagen synthesis, HGF stimulation leads to elevated miR-29 expression, but to repression of collagen synthesis.

However, overexpression of miR-29 in myofibroblastic HSC did not affect the expression of SMA (Figure S1 D).

0024568.g008 Figure 8 miR-29a expression in HSC after 3 days (3d) of primary culture (A) and miR-29a and miR-29b expression in myofibroblastic HSC-T6 (B).

miR-29a expression in HSC after 3 days (3d) of primary culture (A) and miR-29a and miR-29b expression in myofibroblastic HSC-T6 (B).

Together, these data demonstrated that miR-29 specifically inhibits transcription and protein expression of collagen I and IV.

After demonstrating that repression of the collagen-inhibiting miR-29 is an important downstream TGF–β effect, we studied if miR-29 overexpression can overcome the profibrogenic features mediated by TGF-β such as col1A1 induction.

Interestingly, the expression of mature miR-29a, but not of miR-29b, was contrary to the collagen expression during myofibroblastic transition.

TGF-β treatment resulted in highly increased col1A1 expression in HSC cells treated with scrambled miRNA or with antago-miR-29, but only a moderate col1A1 induction in miR-29 overexpressing HSC after transfection with ago-miR-29.

Therefore, miR-29 expression appears to be reciprocally regulated by profibrogenic (TGF-β) and antifibrogenic growth factors (HGF) in HSC, suggesting that miR-29 occupies a central role in responding to the antagonistic actions of HGF and TGF-β in regulating collagen synthesis in activated and transdifferentiated HSC.

miR-29a and miR-29b are downregulated during experimental liver fibrosis in rat.

In agreement with the data on rat HSC, miR-29 is repressed by TGF-β, but upregulated by HGF (Figure S2).

Consistent with the antifibrogenic and inhibitory function of miR-29 in collagen I and IV synthesis and the increase of col1A2 and col4A2 (Figure 7 B), we observed a significant reduction in hepatic miR-29a and miR-29b levels in this experimental BDO mo del (Figure 7 C).

While miR-29 and HGF expression was significantly reduced (C,D), hydroxyproline and collagen mRNA were signifcantly increased in BDO livers (B) (*: p<0.05 **:p<0.01; ***: p<0.001).

Since miR-29 is thought to be involved in modulating expression of extracellular matrix components (Table S3) [30], [31], [32], [34], this makes them promising candidates for collagen I and IV repression after HGF stimulation of HSC.

Keeping with the antifibrotic function of miR-29, miR-29 is reduced in liver biopsies after liver intoxication in mice and after chronic liver disease in humans [35].

Stimulation of primary HSC and myofibroblastic HSC-T6 cells with TGF-β decreased significantly the expression of miR-29a and miR-29b in vitro (Figure 8).

Figure S2 miR-29 expression in human skin fibroblasts after stimulation with HGF.

Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.

In order to analyze if other features of myofibroblastic transition were affected by miR-29, we determined SMA expression in miR-29 treated HSC, because increased SMA assembly is one of the most important features of myofibroblastic transition (Figure S1 A).

Diminished effect of TGF-β on col1A1 expression in miR-29 treated HSC.

Overexpression of miR-29 can overcome TGF-β mediated induction of col1A1.

Table S3 Ranking list of putative collagen targets from miR-29*.

Therefore, in addition to collagen-1, the collagen-4 mRNA could be an important target of miR-29 in HSC after HGF stimulation.

Next, we induced experimental fibrosis by bile duct occlusion (BDO) in rats and studied the miRNA-29 expression during liver fibrogenesis.

Hepatology 36 Mott JL Kurita S Cazanave SC Bronk SF Werneburg NW 2010 Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB.

Thus, our findings convincingly demonstrate that HGF mediates antifibrotic signals by influencing miR-29 expression and thereby counteracting the profibrotic activity of TGF-β.

Reciprocal expression of the collagen subunits, col1A1 and col4A2, and primary and mature miR-29.

Contrary effects of HGF and TGF-β on miR-29 expression in HSC.

Analysis of the transcriptional levels of the primary precursor miRNA (pri-miRNA) revealed that pri-miR-29b/c transcript levels are only moderately suppressed, but that the miR-29a/b gene of chromosome 7 undergoes predominant transcriptional repression upon myofibroblastic transition of HSC.

Furthermore, transfection of ago-miR-29a and ago-miR-29b into HSC suppressed transcription and protein synthesis of collagen type I and IV ((Figure 6A, B).

Insertion of the miR-29 binding sites (wt), but not with mutated binding site (mu), resulted in reduced reporter gene expression by miR-29a treated HSC (A–D).

Among the putative miR-29 binding sites of the collagen mRNA (shown in the Table S4), the following sites were chosen for our further analyses, due to the suggestions of Bartel et al. [50] to function most likely as an inhibitory miR-29 interaction sequence: the region of positon 29–35 in the col4A1 3′-UTR, of postion 404–410 in the col4A5, positon 903–909 in the col1A1, and of position 506–512 in the col1A2 3′-UTR (Table S4).

miR-29 synthesis in HSC is suppressed by TGF-β, but promoted by HGF.

Reduced miR-29a and miR-29b expression in livers after BDO.

In the present study, we now collect evidence that in response to the counteracting HGF / TGF-β signals the miR-29 levels in HSC are contrarily regulated.

To demonstrate the specificity of miR-29 for the binding sites, in the 3′-UTRs two point mutations were incorporated to abolish the putative miR-29 recognition sequences of the collagen-4 mRNA (col4A1, col4A5) and collagen-1 transcripts (col1A1 and col1A2).

These findings are in agreement with the data of Du et al. [33] and recent reports showing the miR-29 regulation of elastin, fibullin and collagen I synthesis [30], [31], [32], [34], [35].

miR-29a and miR-29b regulate collagen I and IV synthesis in activated HSC.

The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays.

Putative miR-29 binding sites in the collagen col1A1 (A), col1A2 (B), col4A1 (C) and col4A5 (D) 3′-UTR (wt) and the corresponding mutated sequences (mu) carrying two point mutations (bold and underlined) were cloned into psiCHECK [TM]-2 vector.

Transfection of HSC-T6 with ago-miR-29a or ago-miR-29b did not result in altered SMA expression when compared to scrambled miRNA treated HSC-T6 cells.

0024568.g005 Figure 5Putative miR-29 binding sites in the collagen col1A1 (A), col1A2 (B), col4A1 (C) and col4A5 (D) 3′-UTR (wt) and the corresponding mutated sequences (mu) carrying two point mutations (bold and underlined) were cloned into psiCHECK [TM]-2 vector.

Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats.

Strikingly, all of the collagen-1 and -4 transcripts contained binding sites for members of the miRNA-29 family in their 3′UTR (Table 1).

Furthermore, our in vitro and in vivo studies on HSC or on BDO -treated fibrotic livers, respectively, suggest that the loss of miR-29 in HSC after TGF-β exposure and during liver fibrogenesis leads to the abolishment of collagen type I and IV repression.

For this purpose, HSC-T6 cells were transfected with miR-29a mimics (ago-miR29a) in comparison to scrambled or miR-29-silencing miRNA (antago-miR-29).

We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.

Recently, miR-29 has been reported to be involved in ECM synthesis.

0024568.g006 Figure 6 mRNA quantification of collagen subunits in HSC treated either with miR-29a, miR-29b (ago-miR-29a, ago-miR-29b), or scrambled miRNA by Real-Time PCR (A).

0024568.g009 Figure 9 The myofibroblastic HSC-T6 cells were transfected with scrambled miRNA, miR-29 mimic (ago-miR-29), or with a miR-29 silencer (antago-miR-29).

miR-29 interaction with the 3′-UTR of col4A1 and col4A5 transcripts.

The synthesis of extracellular matrix proteins is modulated by microRNA-29 (miR-29) in extrahepatic tissue [30], [31], [32], [33].

0024568.g003 Figure 3 RNA of primary HSC in the quiescent stage (day 3 of primary culture) and after myofibroblastic activation (day 7 of primary culture) was analyzed for mRNA col1A2 and col4A1 levels (A), as wells as for the levels of mature miR-29a and miR-29b (B), and the primary transcripts of the miR-29a/b and miR-29b/c gene (C).

This loss of the miR-29a and miR-29b in fibrotic BDO treated livers is attended by reduced levels of HGF upon fibrosis (Figure 7 D).

Repression of collagen synthesis by miR-29a and miR-29b.

The genes for miR-29a and miR-29b [1] are both located on chromosome 7, whereas the genes for miR-29c and miR-29b [2] are located on chromosome 1. Each gene pair is transcribed in tandem resulting in a common pri-miRNA from which the mature miR-29 members are released after further processing [36], [37].

The miR-29 family consists of miR-29a, miR-29b (b [1], b [2]), and miR-29c, which differ in only two or three nucleotides, respectively.

Thus, our data provide detailed evidence for the antifibrotic action of miR-29 in response to HGF signalling that is counteracted by the profibrotic growth factor TGF-β.

Recent reports suggest that miR-29 is also involved in the synthesis of collagen type I in liver fibrosis [34], [35].

mRNA quantification of collagen subunits in HSC treated either with miR-29a, miR-29b (ago-miR-29a, ago-miR-29b), or scrambled miRNA by Real-Time PCR (A).

In order to study miR-29 function in collagen synthesis, we inserted the 3′-UTR sequences downstream of a luciferase reporter (Figure 4 A).

Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro.

The myofibroblastic HSC-T6 cells were transfected with scrambled miRNA, miR-29 mimic (ago-miR-29), or with a miR-29 silencer (antago-miR-29).

The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29 -mimicks by Real-Time PCR and immunoblotting.

We demonstrate that miR-29 is not only involved in collagen type I but also in type IV synthesis of myofibroblastic HSC.

The 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29.

RNA of primary HSC in the quiescent stage (day 3 of primary culture) and after myofibroblastic activation (day 7 of primary culture) was analyzed for mRNA col1A2 and col4A1 levels (A), as wells as for the levels of mature miR-29a and miR-29b (B), and the primary transcripts of the miR-29a/b and miR-29b/c gene (C).

The importance of miR-29 in hepatic collagen homeostasis is underlined by our in vivo data that shows the lack of miR-29 in severe experimental fibrosis after bile duct obstruction.

This loss of miR-29 is suggested to be due to the response of HSC to exposure to profibrogenic mediators as shown by our in vitro findings on TGF-β stimulated HSC.

The level of miR-29 transfected into HSC-T6 cells was analyzed by Real-Time PCR, demonstrating stable levels between 8 h and 48 h post-transfection.

miRNA mimicking miR-29a, miR-29b and a scrambled miRNA control were obtained from Dharmacon (Lafayette, USA).

In this respect, the members of the miR-29 family are the most promising candidates because they are repressed during myofibroblastic transition and they hold highly conserved binding sites in the 3′-UTR of the various subunits of collagen 1 and 4 (Table 1).

The reduced levels of miR-29 during fibrosis are associated with an increase of extracellular miR-29 in serum depending on the fibrotic stage (manuscript in preparation).

Table S4 Putative binding sites of the members of the miR-29 family to the 3′-UTR of different collagens.

Additionally, miR-29a and miR-29b levels (C) and HGF and c-met transcripts (D) were quantified by Real-Time PCR.

8

As shown in Figure 1E and 1F, lovastatin upregulated both miR-29b and p-AMPK expressional levels in cells treated with vehicle (DMSO), but not in cells treated with compound C, demonstrating that lovastatin via AMPK activation upregulated miR-29b expression in HUVECs.

As present in Figure 8B, lovastatin increases miR-29b expression to downregulate PA200 protein, resulting in proteasome inactivation and subsequent suppression of oxidative stress.

In vivo analysis revealed that administration of lovastatin remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats with hyperglycemia, dyslipidemia, and hyperhomocysteinemia, as well as increased miR-29b expressions, reduced PA200 protein levels, and suppression of proteasome activity in aortic tissues.

Lovastatin via upregulation of miR-29b suppresses oxidative stress in endothelial cells treated with oxidized low density lipoprotein (ox-LDL), hydrogen peroxide (H2O2), tumor necrosis factor alpha (TNFα), homocysteine thiolactone (HTL) or high glucose (HG).

However, the downregulation of PA200 induced by lovastatin was abolished by anti-miR-29b and further amplified by overexpression of pre-miR-29b in HUVECs (Figure 2C), suggesting that a decreased PA200 by lovastatin may attribute to miR-29b.

Upregulation of miR-29b expression is a common mechanism contributing to endothelial dysfunction induced by multiple cardiovascular risk factors through PA200 -dependent proteasome -mediated oxidative stress, which is prevented by lovastatin.

In sum, these data suggest that upregulation of miR-29b by lovastatin suppresses oxidative stress in endothelial cells.

Thus, we hypothesized that lovastatin via AMPK activation upregulates miR-29b expression in endothelial cells.

Furthermore, miR-29b replacement inhibits proteasomes in myeloma cells by targeting PSME4 that encodesPA200 [10].

The role of PA200 in lovastatin -induced miR29b -mediated inhibitory effects on proteasome activity was further confirmed by decreasing the expression of PA200.

In this study, we have defined a common mechanism of lovastatin to prevent endothelial dysfunction in different mo dels of CVD, in which lovastatin-increased miR-29b expression is linked to the suppression of oxidative stress, a key step to cause endothelial dysfunction in CVD.

By performing Target Scan, we predicted the 3′-UTR of PSME4 as a miR-29b target (Figure 2B).

Lovastatin inhibited oxidative stress induced by multiple oxidants including ox-LDL, H [2]O [2], TNFa, homocysteine thiolactone (HTL), and high glucose (HG), which were reversed by inhibition of miR-29b in HUVECs.

Although statins have been identified to inhibit proteasome activity in many kinds of mammalian cells [12, 14, 22], including endothelial cells [11], we exploited a novel mechanism in which lovastatin increases miR-29b expression to prevent endothelial dysfunction.

The suppression of lovastatin on proteasome activity was further enhanced if cells were overexpressed with miR-29b.

Anti-miR-29b or overexpression of PA200 abolished lovastatin -induced inhibition of proteasome activity in HUVECs.

This indicates that the reduction of proteasome activity induced by lovastatin is mainly due to upregulation of miR-29b in endothelial cells.

PA200 is a target of miR-29b to regulate proteasome activity in lovastatin -treated endothelial cells.

Thus, it is interesting to establish if statin via regulation of miR-29b inhibits proteasome activity in CVD.

Thus, it was interesting to evaluate if lovastatin via upregulation of miR-29b reduces PA200 gene expression in endothelial cells.

Importantly, these suppressions were abolished in cells infected with anti-miR-29b lentivirus.

These observations indicate that lovastatin increases miR-29b expression in endothelial cells, which is in time- or dose -dependent manner.

In the present study, we firstly reported that lovastatin increases miR-29b expression to prevent endothelial dysfunction.

Anti-miR-29b, as an inhibitor of miR-29b, increased proteasome activity, which is opposite to lovastatin.

Collectively, these data suggest that PA200 mediates the inhibition of proteasome activity by miR-29b in lovastatin -treated cells.

The major finding in this paper is that lovastatin increases miR-29b expression in vascular endothelial cells, which mediates the beneficial effects of lovastatin in CVD.

PA200 is a target of miR-29b in endothelial cells treated with lovastatin.

Lovastatin increases miR-29b expression and decreases proteasome activity in endothelial cells, which is in time/dose -dependent manner.

Lovastatin time/dose -dependently increased miR-29b expression and decreased proteasome activity in cultured human umbilical vein endothelial cells (HUVECs).

Blockage of miR-29b abolishes the effects of lovastatin on suppressing ROS productions induced by multiple cardiovascular risk factors.

Increasing concentrations of lovastatin on (5-50 μM) further enhanced miR-29b expression.

In Figure 1B, lovastatin at 1 μM significantly enhanced miR-29b expression.

PA200 has been identified as a target of miR-29b through PSME4 gene that encodes [10].

To investigate whether miR-29b mediates the suppression of lovastatin on proteasome activity in endothelial cells, HUVECs were infected with lentivirus expressing scrambled microRNA, anti-miR-29b and pre-miR-29b.

The dose -dependent effects of lovastatin on miR-29b expression were next examined in endothelial cells.

Figure 2(A) Cultured HUVECs were infected with lentivirus expressing scrambled microRNA, anti-miR-29b and pre-miR-29b, and then incubated with lovastatin (10 μM) for 24 hours.

Lovastatin increases miR-29b expression in endothelial cells.

MiR-29b mediates lovastatin -induced suppression of proteasome activity in endothelial cells.

To investigate the role of miR-29b in lovastatin -suppressed oxidative stress, we tested the effects of lovastatin in cells deficient of miR-29b.

The levels of p-AMPK in E and miR-29b in F were analyzed by western blot and RT-qPCR, respectively.

Here we report that lovastatin increases miR-29b, resulting in reduction of PA200 and consequent oxidative stress and endothelial dysfunction in rat mo dels of diabetes, dyslipidemia, and hyperhomocysteinemia.

[#] P < 0.05 VS Pre-miR-29b alone group.

Of note, how lovastatin increases miR-29b expression in endothelial cells needs further investigations.

As presented in Figure 2A, lovastatin still dramatically reduced proteasome activity in HUVECs infected with scr-miR lentivirus, but not in endothelial cells infected with anti-miR-29b lentivirus.

However, anti-miR-29b did not alter proteasome activity in HUVECs if PA200 is deficiency by siRNA inference.

Therefore, the current study will open new avenue to investigate the effects of miR-29b in CVD and also provide some insights to drug design for CVD in that targeting miR-29b to improve the outcomes of medical intervention, such as statin.

Lovastatin via AMPK activation increases the levels of miR-29b in endothelial cells.

To investigate whether lovastatin increases miR-29b expression in endothelial cells, we firstly treated cultured HUVECs with varying time-points of lovastatin (10 μM) for 0.5 to 24 hours.

In contrast, pre-miR-29b or PA200 siRNA mimics these effects of lovastatin on proteasome activity.

As shown in Figure 1A, the levels of miR-29b gradually increased beginning from 6 hours and reached peak levels at 24 hours in endothelial cells.

The lentivirus containing scr-miR, anti-miR29b, pre-miR29b, or full length PA200 cDNA, was generated by GenePharma Company (Shanghai, China).

9

From the present study, our results revealed that the expression of Sp1 was significantly increased in high glucose induced rMC-1 cells than MIAT directly targeted miR-29b expression, and MIAT suppression significantly reversed the low expression of miR-29b and high expression of Sp1 induced by high glucose.

Figure 6 miR-29b targets MIAT to regulate its expression(A) TargetScan database predicted that miR-29b has highly conserved target sequence with 3′-UTR of MIAT.

Figure 5The effects of MIAT suppression on the expression of miR-29 and Sp1 in high glucose -induced rMC-1 cells(A) MIAT suppression significantly reversed the decreased expression of miR-29 induced by high glucose.

We revealed that the expression of MIAT was associated with NF-κB (p-p65), NF-κB activated the MIAT, MIAT target regulated miR-29b expression and finally regulated the cell apoptosis.

TargetScan database was used for the online prediction and the results revealed that miR-29b have highly conserved target sequence with MIAT (Figure 6A), the results indicated that MIAT could regulate miR-29b expression.

revealed that MIAT overexpression significantly decreased the expression of miR-29b (Figure 6B), but increased the expression of SP1 (Figure 6C).

MIAT overexpression dramatically decreased the expression of miR-29b (B), while increased the expression of Sp1 in rMC-1 cells (C).

Moreover, miR-29b knockdown significantly reversed the effects of cell apoptosis induced by MIAT suppression, which indicated that the protective function of MIAT suppression was interfered by miR-29b knockdown.

revealed that cell viability was significantly decreased and cell apoptosis was obviously increased by high glucose treatment, then MIAT suppression reversed the effects induced by high glucose, however, miR-29b knockdown significantly reversed the effects induced by MIAT suppression (Figure 7A,B).

MIAT targeted miR-29b to regulate its expressionWe explored the relationship of miR-29b and MIAT.

Our present study showed that MIAT controlled the cell apoptosis in DR might be partly through absorbing miR-29b and inhibiting its function, meanwhile regulating the expression of Sp1.

At the same time, miR-29b inhibited the transcription of Sp1 and then up-regulated its own transcription [36].

MIAT targeted miR-29b to regulate its expression.

Figure 7Interaction of MIAT and miR-29b on high glucose induced rMC-1 cells(A) MIAT suppression significantly reversed the decrease in cell viability induced by high glucose, while miR-29b knockdown significantly reversed the effect induced by MIAT suppression.

miR-29b targets MIAT to regulate its expression.

Previous study investigated that Sp1 expression was directly targeted by miR-29b, which was bound to miR-29b promoter and repressed the expression of miR-29b [35 ].

MIAT suppression increased the expression of miR-29b and SP1.

The effects of MIAT suppression on the expression of miR-29 and Sp1 in high glucose -induced rMC-1 cells.

MIAT suppression increased the expression of miR-29b and SP1In order to explore the potential mechanism between MIAT and cell apoptosis induced by high glucose, miR-29b was selected for further exploration.

The results indicated that MIAT capable of this function might be through harbouring of miR-29b and then regulating the expression of miR-29b and Sp1.

At the same time, miR-29b was differentially expressed in DM [31], however, whether miR-29b regulation plays an important role in DR remains unclear.

The rMC-1 cells in the logarithmic phase were used in the experiment and cultured at 37°C with 5% CO [2] on a 96-well plate, the cells were stimulated by high glucose and transfected with si-MIAT, si-MIAT and miR-29b inhibitor.

miR-29b belongs to the miR-29 family, which acts as a tumour suppressor in many tumour researches.

rMC-1 cells were transfected with si-MIAT and miR-29b inhibitor, then high glucose was used to stimulate the cells.

In order to verify it, Ad-MIAT was constructed and transfected to rMC-1 cells and the expression of miR-29b and its downstream gene SP1 was detected.

The miR-29b inhibitor, si-MIAT and NC were synthesized by Shanghai Yingjun Co.

rMC-1 cells were cultured in a 96-well plate for 24 h, miR-29b inhibitor, si-MIAT, Ad (adenovirus)-MIAT (Ad-MIAT) or their negative control (NC), Ad-carrying GFP (Ad-GFP) transfected the cells by Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

The real-time PCR reflected that when cells were pretreated with si-MIAT and then stimulated by high glucose, the expression of miR-29b was significantly increased than that treated by high glucose only (Figure 5A).

rMC-1 cells transfected with si-MIAT, si-MIAT and miR-29b inhibitor were cultured at 37°C with 5% CO [2] on a 96-well plate for 48 h, and then harvested and stained with propidium iodide (PI) (Sigma) for 30 min.

Study has reported that miR-29b negatively regulated osteoblast differentiation [30].

Interaction of MIAT and miR-29b on high glucose induced rMC-1 cells.

Further clinical therapy based on the NF-κB/MIAT/ miR-29b/Sp1network appears to be important for DR.

Interaction of MIAT, miR-29b and high glucose on cell survival and apoptosisTo identify the effects of MIAT, miR-29b on high glucose induced cell survival and apoptosis.

To identify the effects of MIAT, miR-29b on high glucose induced cell survival and apoptosis.

Interaction of MIAT, miR-29b and high glucose on cell survival and apoptosis.

We explored the relationship of miR-29b and MIAT.

In order to explore the potential mechanism between MIAT and cell apoptosis induced by high glucose, miR-29b was selected for further exploration.

10

This was demonstrated by the data of ChIP which showed a directly targeting miR-29 promotor by p65 and result of cells incubated NF-κB inhibitor, BAY 11-7082 that leaded to abolishment of high glucose -induced upregulation of miR-29 expression.

By luciferase reporter assays and expression analysis of Keap1 in HK [miR−29 mimic] or HK [miR−29 inhibitor] cells, we can draw a conclusion of miR-29 directly regulated Keap1 expression by targeting Keap1 mRNA 3′ UTR.

cell treated with 45 mM + DMSO Detection of abnormal expression of miR-29 and Keap1 in HK-2 cell lead us to determine whether miR-29 directly regulates Keap1 expression.

This finding demonstrated a novel mechanism by which high glucose causes renal tubules injury and may provide insight as to what might be acted as therapy target by providing evidence supporting a role for miR-29 overexpression in the inhibition of Keap1/Nrf2 pathway.

By result of western blotting, we observed that high glucose induced upregulation of Keap1 and downregulation of nuclear Nrf2 was abrogated by miR-29 mimic treatment (Fig.   6b).

a Cells were transfected with miR-29 mimic and b miR-29 inhibitor as well as respective negative control (NC) for 48 h; miR-29 level, translational activity of Keap1 and expression of Keap1 mRNA and protein were determined.

In diabetic rat, miR-29 was downregulated and its expression is negatively correlated with both of serum creatinine and creatinine clearance.

In this present study, we observed that NF-κB activity was enhanced mediated by high glucose induced reduction of deacetylases activity of Sirt1, which leaded to downregulation of miR-29 and inhibition of Keap/Nrf2 signal.

Relative protein band density was quantified by image J. To achieve alteration of expression of miR-29, cells were treated with miR-29 mimic or inhibitor with the negative control (NC) of nonsense strand using Lipofectamine (Invitrogen).

We altered miR-29 expression by cell incubated with miR-29 mimic or inhibitor.

NF-κB was demonstrated to regulate miR-29 expression by directly binding to its promotor.

This data suggested an expressional regulation of miR-29 by NF-κB.

While high glucose induced down regulation of miR-29 contributed to enhancement of Keap1 expression that finally reduced Nrf2 content by ubiquitinating Nrf2.

This was further confirmed by qRT-PCR assay which showed that inhibition of NF-κB pathway by BAY 11-7082 treatment increased miR-29 expression in high glucose-triggered cell.

Fig.  4NF-κB regulates miR-29 expression in high glucose-triggered HK-2 cells.

Additionally, overexpression of miR-29 effectively relieved high glucose-reduced cell viability.

One day later, cells were co -transfected with pGL4-TK-Luc-Keap1 (0.5 μg) with control of pGL-SV40 (internal control, 0.018 μg) and miR-29 mimic or inhibitor.

b Western blot was performed to analyze expression of Keap1 and nuclear Nrf2 in 5.5 mM and 45 mM glucose-triggered HK-2 [miR−29mimic] cells To determine the role of miR-29 on high glucose triggered cell, cell viability was examined in HK-2 [miR−29 mimic] cell.

Calculated Keap1 mRNA expression levels were normalized to the expression levels of GAPDH and relative miR-29 level was normalized to U6 of the same cDNA sample.

Renal tubule was obtained from rats treated with STZ for 4, 8, 12, 16 weeks and lyzed for analysis of miR-29 expression.

Overexpression of miR-29 enhanced cell viability.

Notably, decreased expression of miR-29 by NF-κB signal has been described in cells and tissues [12, 13].

Due to description of decreased expression of miR-29 by NF-κB signal in cells and tissues [12, 13], we speculated that NF-κB/miR-29 axis might be involved in high glucose incubated renal cell.

Level of miR-29 was significantly downregulated in cells incubated with 30 and 45 mM high glucose compared with cell treated with 5.5 mM normal glucose (Fig.   2c).

As shown in Fig.   5b, level of miR-29 declined by 10 fold, binding activity of miR-29 and Keap1 promotor was boosted that leads to increase of the expression of Keap1 mRNA and protein.

We attempted to figure out the target gene for miR-29.

The data showed that high glucose promoted ubiquitination of Nrf2 whereas this promotion was reversed by overexpression of miR-29 (Fig.   6a).

The data of luciferase assay showed that miR-29 directly targets to Keap1 mRNA.

The correlation analysis demonstrated that abnormal miR-29 expression is negatively related to serum creatinine (Spearman correlation is −0.96, P = 0.000; Fig.   1d) and creatinine clearance (Spearman correlation is −0.93, P = 0.000; Fig.   1e).

c miR-29 expression was determined.

Fig.  7Effect of overexpression of miR-29 on cell viability in glucose-triggered HK-2 cell.

b Cells were exposed to BAY 11-7082 for 2 h before 5.5 and 45 mM glucose treatment and miR-29 expression was determined.

MiR-29 directly regulates Keap1.

Fig.  6Ubiquitination of Nrf2 was regulated by miR-29/Keap1 axis in high glucose triggered HK-2 cells.

In HK-2 [miR−29 mimic] cell, level of miR-29 was significantly increased by 12.9 fold, binding activity of miR-29 and Keap1 promotor was reduced and expression of Keap1 mRNA and protein was attenuated compared with pre-NC incubated cell (Fig.   5a).

cell treated with 45 mM + pcDNA was performed to assess whether NF-κB directly binds to miR-29 gene in high glucose cultured HK-2 cell.

The ChIP assay was carried out to detect the possible target miR-29 gene by p65.

All cells were incubated with glucose for 48 h. To evaluate whether NF-κB regulates miR-29 expression in high glucose incubated HK-2 cell, the cells were pretreated with BAY11-7082 (5 μmol/L) (Sigma-Aldrich Co.

These data indicated that miR-29 is involved in pathological process of diabetes and might function as one of modulatory factors.

Renal tubular was obtained for detection of miR-29 level.

As shown in Fig.   7, reduction of cell viability by high glucose was effectively enhanced by miR-29 mimic treatment.

Among these, miR-29 is observed in diabetic patients [10] and also ameliorates hyperglycemia -induced renal dysfunction [11].

Notably, both miR-29 and NF-κB activity play vital roles in high glucose induced decrease of cell viability.

pre-NC or NC To determine the downstream molecule of miR-29/Keap1 axis, we examined Nrf2, a nuclear transcriptional factor that commonly activated by Keap1, in 45 mM high glucose-triggered HK-2 [miR−29 mimic] cell.

cell treated with 45 mM + pcDNA Chip assay was performed to assess whether NF-κB directly binds to miR-29 gene in high glucose cultured HK-2 cell.

The data showed that miR-29 level was decreased in time -dependent manner after rat mo del of diabetes established (Fig.   1c).

Level of microR-29 (miR-29) was assessed using quantitative RT-PCR.

We made a prediction of the possible binding site in Keap1 3′ UTR by miR-29 and this was demonstrated by online bioinformatics analysis (data is not shown).

Thus we speculated that NF-κB/miR-29 axis might be involved in high glucose incubated renal cell.

Acetyl-p65 HK-2 miR-29 Nrf2 Serum creatinine Diabetes mellitus is a common metabolic disorder which is associated with chronic complications such as angiopathy, retinopathy, and peripheral neuropathy.

In summary, our data suggested that high glucose induced renal tubular injury might be a process of a signal transduction pathway of Sirt1/NF-κB/miR-29/Keap1/Nrf2.

Combination of p65 and miR-29 promotor was assessed using chromatin immunoprecipitation.

NF-κB binds to miR-29.

High glucose promotes ubiquitination of Nrf2 via miR-29/Keap1 axis.

As shown in Fig.   4a, high glucose promoted association between p65 and miR-29 promotor in a manner of time dependent.

To assess the correlation between the serum creatinine or blood urea nitrogen (BUN) and serum miR-29 level, the Spearman correlation coefficient was used.

a Cells were stimulated with 5.5 and 45 mM glucose for 12, 24, 48 h and combination of p65 and miR-29 gene was examined using.

Correlated analysis was performed to assess relationship between d miR-29 level and Serum creatinine as well as e miR-29 level and creatinine clearance.

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Mechanism study revealed that carvedilol inhibited ROS -induced Smad3 activation, and Smad3 signal negatively modulated miR-29b expression, miR-29b could efficiently inhibit fibrosis-related genes expression in cardiac fibroblasts.

We showed that carvedilol specifically inhibits expression of the ECM-related proteins Col1a1, Col3a1, and α-SMA and promotes miR-29b expression in infarcted myocardium and cardiac fibroblasts in vitro.

Using Smad3 siRNA and 2 Smad3 inhibitors, SIS-3 and Nar, we demonstrated that Smad3 signaling negatively regulates miR-29b expression derived from only from the miR-29b-2 precursor (Figure 4E, 4H).

ECM-related Col1a1, Col3a1, and α-SMA expression and miR-29b expression in carvedilol -treated rat cardiac fibroblasts.

Enforced expression of miR-29b decreased Col1a1, Col3a1, and α-SMA expression.

control group, N = 4. D. Upregulation of mature miR-29b in smad3 knockdown rat cardiac fibroblasts.

After a 24-h incubation, real-time PCR analysis indicated that the miR-29b mimic inhibited Col1a1, Col3a1, and α-SMA expression at the mRNA level (p < 0.05, p < 0.01, and p < 0.001, respectively) (Figure 5A).

Additionally, de-suppression of miR-29b by smad3 inactivation contributes to ECM-related gene expression.

ECM-related Col1a1, Col3a1, and α-SMA expression and miR-29b expression in the border zone of the infarcted region.

Our results confirm that miR-29b inhibits Col1a1,Col3a1, and α-SMA expression at both the mRNA and protein level (Figure 5A, 5B, S3).

Carvedilol inhibited ROS-activated Smad3 signaling involved in ECM related genes and miR-29b expression.

In this study, we found that miR-29b was upregulated in the border zone of infarcted myocardium in the rat AMI mo del treated with medium- and high-dose carvedilol (Fig. 2C).

ECM-related genes and miR-29b expression in carvedilol -treated rat cardiac fibroblasts in vitro A dose-course study of the effect of carvedilol on Col1a1, Col3a1, and α-SMA expression was demonstrated by quantitative real-time PCR and Western-blotting assay, respectively (Figure 3A, Figure 3B, S2).

miR-29b was also upregulated by carvedilol in rat cardiac fibroblasts in vitro (Fig. 3C).

miR-29b was shown to regulate myocardial fibrosis [14, 16], but whether carvedilol could modulate miR-29b expression was unknown.

Previous studies indicated that miR-29b can regulate the expression of ECM-related genes, including Col1a1, Col1a2, and Col3a1 [16, 38].

Consistent with these results, miR-29b also inhibited Col1a1, Col3a1, and α-SMA protein expression compared to that observed using the scrambled oligonucleotide (p < 0.05 and p < 0.001, respectively).

Taken together, these data demonstrate that inactivation of ROS -induced Smad3 signaling and miR-29b upregulation mediate the anti-fibrotic effect of carvedilol in MI -induced cardiac fibrosis (Figure 6).

Blank group, N = 4. G. Upregulation of mature miR-29b in SIS-3 or Nar -treated rat cardiac fibroblasts.

This result was consistent with previous reports that Smad3 signaling promotes renal fibrosis by inhibiting miR-29 [15, 38].

Smad3 signaling pathway, ECM-related genes and miR-29b expression in rat cardiac fibroblasts.

Figure S3 Col1a1, Col3a1, and α-SMA protein expression in miR-29b -modified rat cardiofibroblasts.

Compared with the control group, mature miR-29b was significantly up-regulated in the 2 µM and 4 µM carvedilol -treated rat cardiac fibroblasts (p < 0.05 and p < 0.05, respectively) (Figure 3C).

miR-29b modulated expression of ECM-related gene in cardiac fibroblasts.

We also confirmed that miR-29b-2 precursor expression was much higher than miR-29b-1 in myocardium (Fig. 2D) and cardiac fibroblasts (Fig. 3D); miR-29b-2, but not miR-29b-1, could be modulated by carvedilol in vivo (Fig. 2D) and in vitro (Fig. 3D).

miR-29b-2 in blank control group, N = 4. To study the expression of ECM-related genes after miR-29b mimic treatment, we transfected rat cardiac fibroblasts with the miR-29b mimic or scrambled oligonucleotide.

In the sham surgery control group, the expression level of the mir-29b-2 precursor was much higher than that of the mir-29b-1 precursor (p < 0.001) (Figure 2D).

ECM-related genes and miR-29b expression in AMI -induced fibrotic myocardium treated with carvedilol.

showed that the expression of the mir-29b-2 precursor was much higher than that of the mir-29b-1 precursor (p < 0.01) (Figure 3D).

ECM-related genes and miR-29b expression in carvedilol -treated rat cardiac fibroblasts in vitro.

Expression of the miR-29b-2 precursor, but not the miR-29b-1 precursor, increased significantly in a dose -dependent manner in carvedilol -treated rat cardiac fibroblasts (p < 0.05) (Figure 3D).

C. Mature miR-29b expression by quantitative real-time PCR assay.

MicroRNA-29b, a regulator of fibrosis [14- 16], is dysregulated in AMI [14].

control group, N = 4. D. miR-29b-1 and miR-29b--2 precursor expression by quantitative real-time PCR assay.

D. miR-29b-1 and miR-29b-2 precursor expression by quantitative real-time PCR assay.

Methods for coding gene and miR-29b precursor expression detection were as follows: First-strand cDNAs were synthesized using a mixture of oligo (dT) 15 and random primers with Superscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA).

The fibrosis regulator, miR-29b, was validated to be modulated by carvedilol, contributing to the anti-fibrotic effect of carvedilol.

miR-29b-1 in control group; * p < 0.05 vs.

The quantity of mature mir-29b and miR-29b-2 precursors, but not miR-29b-1 precursor, increased significantly in SIS-3 or/naringenin -treated rat cardiac fibroblasts (p < 0.05 and p < 0.01, respectively) (Figure 4G,4H).

miR-29b-1 in blank control group, * p < 0.05, ** p < 0.01 vs.

The quantity of mature mir-29b was significantly higher in Smad3 siRNA -modified rat cardiac fibroblasts (p<0.05) (Figure 4D).

Consistent with this result, the quantity of miR-29b-2 precursor, but not miR-29b-1 precursor, was also dramatically higher in Smad3 siRNA -modified rat cardiac fibroblasts (p < 0.01) (Figure 4E).

Mature rat miR-29b can be derived from miR-29b-1 and miR-29b-2 precursors, which are transcribed from two different loci in the rat genome (www.

Methods for mature miR-29b level detection were as follows: the total RNA was used to detect mature miRNA level using Bulge-Loop miRNA qRT-PCR kit (Guangzhou Ribobio, China).

The quantity of mature miR-29b in the AMI border zone was significantly higher in the CAR-M and CAR-H groups (p < 0.05 and p < 0.01, respectively) than in the untreated AMI group (Figure 2C).

miR-29b-1 precursor, * p < 0.05 vs.

miR-29b-1 in control group, [#] p < 0.01 vs.

In this study, we assessed the hypothesis that Smad3 signaling and miR-29b mediate the effect of carvedilol on attenuating AMI -induced myocardial fibrosis in rat.

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Therefore, we concluded that CA might target miR-29b-3p to inhibit HMGB1 expression in BDL -induced liver fibrosis.

FIGURE 6Carnosic acid inhibits HMGB1 expression and LX2 activation by enhancing miR-29b-3p.

As shown in Figures 5B,C, mimic-miR-29b-3p significantly decreased HMGB1 protein expression, whereas mimic-miR-300 had no effect on HMGB1 protein expression in LX2 cells.

In addition, the expression of α-SMA in LX2 cells was inhibited by mimic-miR-29b-3p.

We then explored this possibility in vitro and found that miR-29b-3p was up-regulated in LX2 cells after CA treatment.

Among the 25 down-regulated miRNAs, miR-300 and miR-29b-3p were predicted to bind to the 3′-UTR of HMGB1 mRNA.

These results confirmed that HMGB1 is a direct target of miR-29b-3p.

Therefore, we hypothesized that CA -induced protection against BDL -induced liver fibrosis occurred through miR-29b-3p up-regulation.

Regulatory Effect of miR-29b-3p on HMGB1 Expression.

These data suggest that miR-29b-3p might play a key role in the control of HMGB1 expression.

The results showed that CA decreased HMGB1 expression and LX2 activation at least partially in an miR-29b-3p -dependent manner.

Therefore, CA increases miR-29b-3p to inhibit the HMGB1/TLR4/NF-κB pathway, thereby attenuating BDL -induced liver fibrosis.

We subsequently transfected LX2 cells with the miR-29b-3p antagomir in the presence or absence of CA treatment and assessed the expression levels of HMGB1, TLR4, and α-SMA and the translocation of p65 to the nucleus.

microRNA-29b prevents liver fibrosis by attenuating hepatic stellate cell activation and inducing apoptosis through targeting PI3K/AKT pathway.

We predicted that miR-300 and miR-29b-3p bind to the 3′-UTR of HMGB1 mRNA and found that the expression of miR-300 and miR-29b-3p in the liver was consistent with that obtained in a previous report.

As shown in Figures 5A, 6D, the in vivo, miR-29b-3p expression was significantly increased by CA treatment, and this increase was positively associated with the protective effect of CA in BDL rats.

Suppression of hepatic stellate cell activation by microRNA-29b.

To investigate the effects of miR-29b-3p over -expression on the activation of HSCs, α-SMA expression in LX2 cells was determined by western blot.

For further studies on the effects of CA on HMGB1 in cells, we detected the expression of miR-29b-3p by qPCR.

Moreover, the luciferase activities of the mutated HMGB1 3′-UTR and the empty vector were not inhibited by the miR-29b-3p mimic (Figure 6C).

Based on the miRNA database and the results of our experiment, we determined the effect of mimic-miR-300 and mimic-miR-29b-3p on the expression of HMGB1 in LX2 cells.

The results revealed that the level of α-SMA was decreased by treatment with mimic-miR-29b-3p (Figure 6A), indicating that miR-29b-3p can inhibit the activation of LX2 cells.

CA Reduces HMGB1 Expression and LX2 Activation by Enhancing miR-29b-3p.

Importantly, the miR-29b-3p antagomir could reverse the CA -mediated inhibition of the HMGB1/TLR4/NF-κB pathway and α-SMA levels.

Based on the miRNA database and our preliminary experiment, we found that miR-29b-3p might play a key role in control of HMGB1 expression in BDL -induced liver fibrosis.

The main objectives of this study were as follows: (1) to elucidate the role of the antifibrosis effect of CA in protecting against BDL; (2) to test whether the activity of CA against liver fibrosis is associated with the signaling mechanisms of HMGB1/TLR4/NF-κB pathway in BDL; and (3) to investigate whether CA inhibits HMGB1/TLR4/NF-κB by promoting the expression of miR-29b-3p.

Given that HMGB1 was predicted to be a putative target of miR-29b-3p (Figure 6B), the protein levels of HMGB1 were found to be decreased by mimic-miR-29b-3p (Figure 5B).

Luciferase assays also confirmed that HMGB1 is a target of miR-29b-3p.

MicroRNA-29b-3p prevents Schistosoma japonicum- induced liver fibrosis by targeting COL1A1 and COL3A1.

In addition, decreased HMGB1 expression was observed in the mimic-miR-29b-3p group compared with that in the control group.

The animal experiments revealed that CA treatment could significantly alleviate BDL -induced liver fibrosis in rats and reverse the decrease in miR-29b-3p.

For functional analyses, LX2 cells were transfected with antago-miR-29b-3p or antago-miR-29b-3p control.

The plasmid and the miR-29b-3p mimic or the miR-29b-3p negative mimic were co -transfected into LX2 cells.

Dual-luciferase reporter plasmids of miR-29b-3p-HMGB1 were purchased from GenePharma Corp.

In summary, our data indicate that the miR-29b-3p/HMGB1/TLR4/NF-κB signaling pathway might be essential for liver fibrosis and that CA could be a promising therapeutic agent in liver fibrosis by modulating the miR-29b-3p/HMGB1/TLR4 signaling pathway.

Transfected experiments were performed using 2 μg pcDNA3.1/HMGB1, 50 nM mimic-miR-29b-3p, 50 nM mimic-miR-300 or 50 nM antagomiR-29b-3p (GenePharma) and Lipofectamine 3000 (Invitrogen, United States) according to the manufacturer’s instructions.

The construct was cotransfected into LX2 cells with the miR-29b-3p mimic or the miRNA negative control (con -mimic).

We subsequently discovered that the protein level of HMGB1 decreased only following treatment with the miR-29b-3p mimic.

We then generated an HMGB1 3′-UTR luciferase reporter containing the miR-29b-3p -binding sites (HMGB1 wild-type 3′-UTR) or mutated sites (HMGB1 Mut 3′-UTR).

The qPCR results indicated that the miR-300 and miR-29b-3p levels were significantly lower (P < 0.01) in the BDL rats than in the controls (Figure 5A).

13

Although there are presumably additional targets regulated by miR-29 and miR-203, our experiments suggest that VEGFA suppression is mediated, at least in part, by overexpression of these miRNAs.

Overexpression of miR-29b was detected on days 3 and 5. Expression level of miR-203 was also markedly higher on days 3 and 5 (4.2-fold and 2.6-fold, respectively) (Figure 3B).

Relative expression levels of miR-29 family members (A) and miR-203 (B) in isolated (ISO) rats are in comparison to control levels expressed as 1 on the graph.

These data suggest that overexpression of miR-29 and miR-203 contributes to isolation -induced delays of wound healing, partially by suppressing VEGFA.

0072359.g004 Figure 4(A) The predicted miR-203 and miR-29 targeting sequences located in the 3’-untranslated region (3’-UTR) of VEGFA mRNA.

Cells were co -transfected with constructs containing the predicted targeting sequence (WT) or mutated targeting sequence (Mutant) cloned into the 3’-UTR of the reporter gene, along with miRNA mimics of miR-29 or miR-203.

Based on bioinformatics analysis, a highly conserved miR-29 isoform -targeting sequence and a miR-203 -targeting sequence were identified in the VEGFA mRNA 3’ UTR (Figure 4A).

MiR-203 and miR-29 directly target VEGFA mRNA.

Expression of miR-29 family members, miR-203 and SOCS3 mRNA in wounded tissues by qRT-PCR.

In this study, VEGFA was the only healing -associated mRNA targeted by miR-29 and miR-203 (out of the nine that were tested).

0072359.g003 Figure 3 Expression of miR-29 family members, miR-203 and SOCS3 mRNA in wounded tissues by.

In accordance with these findings, our results demonstrate that miR-29 family members and miR-203 were persistently overexpressed across healing in isolated rats.

For functional analysis, miR-29 (a, b, c) mimics, miR-203 mimics and non -targeting miRNA mimics (Dharmacon, Lafayette, CO, USA) were transfected into cells using DharmaFECT Transfection Reagent 1 (Dharmacon) per the manufacturer’s instructions.

Reduced expression of miR-29 family members was recently reported in different fibrotic organs [31- 33].

MiR-29 is a typical multifunctional miRNA which is involved in regulating the epithelial-mesenchymal transition, cellular differentiation, extracellular matrix remo deling, and angiogenesis [29, 30].

Mutation of the putative miR-203 binding site abolished the effect of miR-203 while leaving the action of all three miR-29 isoforms unaffected (B).

Similarly, mutation of the putative miR-29 site led to abolishing the action of the miR-29 isoforms (Figure 4C).

Intriguingly, both miR-29 and miR-203 are important regulators of wound-specific cell functions and the cytokine network [38, 39].

Similarly, mutation of the putative miR-29 binding site abolished the action of the miR-29 isoforms (C).

As expected, mutation of the putative miR-203 binding site abolished the effect of miR-203 while leaving the action of all three miR-29 isoforms unaffected (Figure 4B).

In this study, we hypothesized that social isolation delays oral mucosal wound healing, and healing -associated genes and miRNAs (i. e., miR-29 and miR-203) play a role in this process.

Isolation Stress and Levels of miR-29 and miR-203.

Considering the important role of miR-29 and miR-203 in both dermal and mucosal repair, these results suggest a putative mechanism for isolation -induced healing impairments through the modulation of VEGFA.

This suggests there are novel roles of miR-29 and miR-203 in isolation-impaired healing.

Intriguingly, the isolated rats persistently exhibited higher levels of miR-29 family members and miR-203.

To test this, we selected and determined the levels of two miRNAs in wounds: miR-29 and miR-203.

In both the putative miR-29 and miR-203 binding sites, the 6 to 8 nucleotides of the “seed region” were replaced with a restriction enzyme site with minimal complementarity to the miRNA sequence (see Materials and).

14

At 2 h, we saw a trend of upregulation of miR-29b in neurons that steadily increased up to 4-fold at 24 h. In contrast, in astrocytes, miR-29b showed a trend of upregulation later, after 6 h and a significant level increase by 2-fold only after 12 h. MiR-30b, miR-107 and miR-137 were uniquely upregulated only in astrocytes at different time-points, starting 6 h after OGD event.

Park and colleagues [69] have shown that family members of miR-29 upregulate the expression of p53 through regulation of transcription of cdc42 and p85α in tumor cells and as such induce apoptosis.

Furthermore, we show that a known neuroprotectant,, can reduce the expression of miR-29b, which is highly upregulated in ischemic neurons.

In this study we were able to show a novel effect of IGF-I, that of suppression of miR-29b expression in neurons.

MiR-29b expression was upregulated in astrocytes as well, but to a lesser magnitude than in neurons.

We speculate that the downregulation of miR-29b by IGF-I could possibly play a role in the anti-apoptotic signaling afforded by activation of the IGF-I receptor, but further studies would be needed in order to substantiate this hypothesis.

A direct comparison of expression levels of all the miRNAs used for our study between neurons and astrocytes undergoing OGD conditions, showed a significant difference at different time-points only for miR-29b, miR-30b, miR-107 and miR-137 (Figure 1).

In his study, Dharap [28] reported miR-29b (at 3, 6 and 24 h) and miR-137 (at 24 h) to be downregulated in a MCAo adult rat mo del, while Lei [33] reported the same findings in a TBI adult rat mo del at 6 h post injury.

MiR-29b, miR-30b, miR-107 and miR-137 showed a significant difference in their expression levels between neurons and astrocytes undergoing OGD conditions (Figure 1).

We screened the same miRNAs as described above (miR-21, miR-29b, miR-30b, miR-107, miR-137, miR-210) and found that IGF-I significantly decreases the expression of mir-29b in neurons.

Another recent study found that miR-29 family could regulate DNA methylation via DNA methyltransferase 3A and 3B transcriptional regulation [71].

Garzon and colleagues [70] reported that over -expression of synthetic miR-29b in acute myeloid leukemia (AML) cell lines and primary AML blasts induced apoptosis.

MiR-29b is upregulated in both neurons and astrocytes post-OGD, but at different time-points and with remarkable difference in magnitude.

MiR-29b showed a trend of upregulation at 6 h post-OGD with >1.7-fold increase, and >2-fold at 12 h post-OGD (p<0.05).

These studies strongly support the idea that by the blockage or enhancement of miR-29b expression we could influence apoptosis of cells of interest [69], [70].

Our findings that miR-29b is upregulated in astrocytes and neurons after ischemia in vitro opens the path for further investigations to the role of miR-29 in apoptosis in other forms of dementia that have been related to lacunar brain ischemia.

MiR-29b showed the highest expression alteration in neurons exposed to OGD.

Insulin-Like Growth Factor I (IGF-I) temporally decreases microRNA-29b.

We screened the same miRNAs as described above (miR-21, miR-29b, miR-30b, miR-107, miR-137, miR-210).

0014724.g003 Figure 3 temporally decreases microRNA-29b.

15

The Mir29b‐1 gene was targeted for mutation using custom‐made Transcriptional Activator‐Like Effector Nucleases (TALENs, Cellectis Bioresearch) which targeted the sequence TTTAAATAGT GATTGTC tagcaccatttgaaa TCAGTGTTCTTGGTGGA where each TALEN monomer binds the target sequences underlined on opposite strands, separated by a spacer (lowercase).

Neuromodulin (Gap43), another predicted target of miR‐29 that can reduce eNOS activation by inhibiting calcium/calmodulin, was up‐regulated from 2.9 FPKM in wild‐type rats to 6.8 FPKM in Mir29b‐1/a [−/−] rats (P = 0.04) according to the RNA‐seq analysis of gluteal arterioles (Dataset EV3).

Figure 4miR‐29 regulates genes involved in determining NO levels, including Lypla1 Mir29b‐1/a mutation in rats led to differential expression of several genes relevant to the regulation of NO bioavailability in gluteal arterioles.

The expression profiles of 18,615 detected genes correctly clustered the samples by genotype, indicating the Mir29b‐1/a mutation had substantial and reproducible effects on the gene expression profile in gluteal arterioles (Appendix Fig S7).

Of the 12 predicted miR‐29 target genes with known involvement in NO regulation (Dataset EV2), lysophospholipase I (Lypla1) was up‐regulated from 65 FPKM in wild‐type rats to 104 FPKM in Mir29b‐1/a [−/−] rats (P = 0.005, adjusted P = 0.05; Fig  4A).

Mir29b‐1/a mutation in rats led to differential expression of several genes relevant to the regulation of NO bioavailability in gluteal arterioles.

Intraluminal delivery of anti‐miR‐29b‐3p in arterioles from non‐ DM human subjects or rats or targeted mutation of Mir29b‐1/a gene in rats led to impaired EDVD and exacerbation of hypertension in the rats.

Of the 286 genes relevant to NO regulation (Dataset EV2), 179 were detected in the RNA‐seq analysis, of which 32 were differentially expressed between Mir29b‐1/a [−/−] and wild‐type littermates (adjusted P < 0.05; Fig  4A).

The mutation of Mir29b‐1/a gene led to preferential differential expression of genes related to nitric oxide including Lypla1.

The residual miR‐29b‐3p might be expressed from the separate Mir29b‐2 gene.

A mutant rat line, SS‐Chr 13BN‐ Mir29b1 [em1Mcwi], hereafter referred to as Mir29b‐1/a mutant or Mir29b‐1/a [−/−] rat, was generated, having a TALEN‐induced 4‐bp deletion within the TALEN target spacer, TTTAAATAGT GATTGTC tagca—ttgaaa TCAGTGTTCTTGGTGGA confirmed by Sanger sequencing and predicted to disrupt the rno‐miR‐29b‐3p sequence.

We used a Transcriptional Activator‐Like Effector Nucleases (TALEN) method to target the Mir29b‐1/a gene on the genetic background of SS‐Chr13 [BN] rats (Geurts et al, 2010).

Taken together, these data indicated the mutant rat, which we designated Mir29b‐1/a [−/−], was a mo del of robust miR‐29a‐3p and miR‐29b‐3p knockdown.

Mir29b‐1/a mutation preferentially influenced genes relevant to the regulation of NO bioavailability.

miR‐29b‐3p mimic increased, while anti‐miR‐29b‐3p or Mir29b‐1/a gene mutation decreased, nitric oxide levels in arterioles.

The genes shown were differentially expressed between Mir29b‐1/a [−/−] rats (KO) and wild‐type (WT) littermates with adjusted P‐values < 0.05.

Real‐time PCR analysis did not reproducibly detect Gap43 mRNA in the small amount of gluteal arteriole samples but confirmed Gap43 mRNA was up‐regulated in the carotid artery of Mir29b‐1/a [−/−] rats (Appendix Fig S8).

The development of hypertension was significantly exacerbated in Mir29b‐1/a [−/−] rats.

miR‐29b is encoded by Mir29b‐1 and Mir29b‐2 genes.

Four nucleotides overlapping with the seed region of miR‐29b‐3p were deleted in the SS‐Chr 13BN‐ Mir29b1 [em1Mcwi] (i. e., Mir29b‐1/a mutant or Mir29b‐1/a [−/−]) rat.

Abundance of miR‐29b‐3p and miR‐29a‐3p in the EC elute from the gluteal arterioles of Mir29b‐1/a [−/−] rats.

We developed Mir29b‐1/a mutant rats to further examine the role of miR‐29 in normal endothelial function.

We identified and established a colony of rats with deletion of four base pairs in the genomic segment of the Mir29b‐1/a gene that encodes nucleotides 6–9 in the sequence of mature miR‐29b‐3p (Fig  2C).

Treatment with Lypla1 si RNA improved EDVD in arterioles obtained from T2 DM patients or Mir29b‐1/a mutant rats or treated with anti‐miR‐29b‐3p.

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miR-29b is a key repressor of renal and pulmonary fibrosis [56, 57] and has recently been shown to inhibit hepatic fibrogenesis by directly targeting PIK3R1 and AKT3 in hepatic stellate cells [58].

The miRNAs with the most consistent female-biased expression were miR-29b (Fig.   3) and miR-152 (Fig.   3 and Fig.   4) with both showing an approximate 2-fold higher expression in females than males at 15, 21, 52, and 78 weeks of age.

The miR-29 family members are induced by estrogen and reduce fibrosis by inhibiting expression of collagens [43].

Despite only seven sexually dimorphic miRNAs being in common between the adult and the old rats (miR-125b-5p, miR-152, miR-29b, miR-374, miR-96, miR-99a*, and miR-99a), 91% (74/81) of the mRNA targets in the old rats were also mRNA targets in the adult rats.

The role of the miR-29 family, including miR-29b, in fibrosis disease has been recently reviewed and additional targets appear to be collagens [59].

In addition, the miR-29 family members appear to play a significant role in the development of liver fibrosis, possibly through the regulation of collagen expression [59].

The average log2 relative expression per group of qPCR and microarray data are displayed for miR-29b, miR-34a, miR-96, and miR-154* such that the global average expression for qPCR (n = 5) and microarray data (n = 4 or 5) is equal to zero.

Female-biased expression of miR-29b and miR-152 occurred from 15 to 78 weeks of age while male-biased expression of miR-125b-5p and miR-99a occurred during this same age span.

Measurement of miR-29b expression by microarray showed female-biased expression at 15, 21, 52, and 78 weeks of age.

The expression of six (miR-29a, miR-29b, miR-29c, miR-34a, miR-375, and miR-466b-2*) was altered in both males and females (Table  1).

As rats mature from adults to old-age, miRNAs involved in cell death, cell proliferation, and cell cycle (miR-29 family and miR-34a) were found to change expression.

The miR-29 family and miR-34a, whose expression was altered in both older males and older females, are relatively well-studied miRNAs, and their roles in cell proliferation and apoptosis are established [75, 76].

In addition, miR-29 family members may be involved in susceptibility to fibrosis.

Significant (p < 0.05) sex difference was confirmed by qPCR for miR-29b only at 52 weeks of age (female > male, Fig.   6a).

17

Interestingly, ZL-F had higher cardiac Med13 expression than ZL-M. (d– f) Graphs show qRT-PCR data on the expression of cardiac microRNA miR-29a, b and c. Expression of all miR-29 family miRNAs were increased in ZDF-F and ZDF-M. miR-29c was the most differentially expressed miRNA between male rats (f) whereas miR-29b was the most differentially expressed miRNA between female rats (e).

Differential expression of miR-29b was highest between ZDF-F and ZL-F, while differential expression of cardiac miR-29a and c were prominent between ZDF-M and ZL-M. Collectively our data show that T2DM -associated CVD progression in ZDF-F and ZDF-M is structurally and mechanistically different.

Since increased expression of miR-208a is associated with cardiac hypertrophy, the female-specific increase in cardiac miR-208a expression could have contributed to the increased susceptibility to cardiac hypertrophy in ZDF-F. Increases in the circulating levels of miR-29 family miRNAs in children with T1DM and adults with T2DM 42, 43 emphasize the clinical relevance of this biomarker in DM.

DM -associated dysregulation of miR-208a-MED13 signaling and increase in miR-29 family miRNAs occur in both male and female ZDF rats, however, only ZDF-F rats exhibited myocardial damage indicating that cardiac repair is impaired in ZDF-F. It is conceivable that the higher expression of cardio-reparative Agtr2 in ZL-F compared to ZL-M (Fig.   9a) could have provided increased protection despite the higher expression of pro-hypertrophic miR-208a in ZL-F heart compared to ZL-M heart.

Here we show for the first time that while all miR-29 family miRNAs increased in response to diabetes in both sexes, there was a sex difference in their expression patterns.

Thus, increases in the expression levels of the individual DM -associated miR-29 isoforms are also largely dependent on sex.

Cardiac miR-29 family miRNA expression patterns are different in male and female diabetic rats.

Thus, there were sex differences in miR-29 family miRNA expression.

Cardiac expression of AT2R (Agtr2), Med13, miR-208a, and miR-29 family miRNAs were determined using mRNA and miRNA isolated from frozen heart tissues as described previously [44].

Figure 9Expression patterns of cardioprotective Agtr2 and Med13, and cardio- deleterious miR-208a and diabetic marker miR-29 family miRNAs in heart tissues of 5-month old healthy and diabetic, male and female rats.

Additionally, miR-29b expression was highest in ZDF-F (Fig.   9e).

Cardiac expression of all members of the miR-29 family increased in both ZDF-F and ZDF-M compared to ZL-F and ZL-M (Fig.   9d–f, p < 0.05).

Given the critical role of miR-29 in both DM and cardiac structure, we compared the cardiac expression of miR-29a, b and c between our groups.

miR-29b is implicated in the development of early aortic aneurysm [68], whereas miR-29c is considered as a signature molecule of hyperglycemia [69].

We previously showed that increased miR-29 family miRNAs correlate with DM -induced cardiomyocyte disorganization [44].

We showed that elevated miR-29 family miRNAs correlate with increased cardiomyocyte disarray in 15-week old ZDF-M [44].

Members of the microRNA miR-29 family (miR-29a, b, and c) serve as mechanistic biomarkers for diabetes (T1DM and T2DM) and cardiovascular damage.

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For instance, during postnatal aortic development, miR-29 was found to be differentially expressed to downregulate elastin [14].

Last but not least, in a very recent study [30], a comparison of miRNA expression profile between the dermis tissues of the young and elderly revealed that, several miRNAs, including miR-29, were upregulated in aged dermis.

In cells haploinsufficient for elastin and in bioengineered vessels, inhibition of miR-29 could increase elastin expression levels [15].

Rats were assigned into six experimental groups (n = 12 each): (1) control, sham-operated rats to serve as healthy control; (2) PFD, vaginal distention was performed on the rats to induce PFD symptoms, and saline was injected 2 weeks after the operation; (3) BMSC (control) + bFGF, PFD rats were injected with control BMSCs and free bFGF 2 weeks after the operation; (4) BMSC (control) + PLGA-bFGF, PFD rats were injected with control BMSCs and bFGF -loaded PLGA 14 days after the operation; (5) BMSC (anti-miR-29) + bFGF, PFD rats were injected with miR-29a-3p -inhibited BMSCs and free bFGF 2 weeks after the operation; (6) BMSC (anti-miR-29) + PLGA-bFGF, PFD rats were injected with miR-29a-3p -inhibited BMSCs and bFGF -loaded PLGA 2 weeks after the operation.

Four different in vitro cultures were established: (1) BMSC (control) + bFGF, control BMSCs with 20 ng/ml free bFGF; (2) BMSC (control) + bFGF-PLGA, control BMSCs with 20 ng/ml equivalent bFGF -loaded PLGA NPs; (3) BMSC (anti-miR-29) + bFGF, miR-29a-3p inhibited BMSCs with 20 ng/ml free bFGF; (4) BMSC (anti-miR-29) + bFGF-PLGA, miR-29a-3p -inhibited BMSCs with 20 ng/ml equivalent bFGF -loaded PLGA NPs.

Downregulation of elastin by miR-29 family miRNAs has been wi dely reported.

from the above studies consistently point to an important role of miR-29, miR-29a-3p in particular, in our current work, in regulating the expression of elastin in the mammalian system, including BMSCs.

Recently in a study performed in vascular smooth muscle cells, miR-29 -mediated elastin downregulation was found to contribute to osteoblastic differentiation induced by inorganic phosphorus [16].

[##] P < 0.01 vs BMSC (control) + bFGF-PLGA and BMSC (anti-miR-29) + bFGF We next established a rat PFD mo del to test the effect of miR-29a-3p -inhibited BMSC transplantation on PFD symptoms in vivo.

Importantly in rats receiving injections of miR-29a-3p -inhibited BMSCs, the decreased void volume and bladder void pressure were both rescued (Fig.   6b and c, fifth and sixth columns), with defects in BMSC (anti-miR-29) + bFGF-PLGA group rats almost completely restored to similar levels of the sham-operated control rats (Fig.   6b and c, sixth column).

[$] P < 0.05 vs PFD, BMSC (control) + bFGF, BMSC (control) + bFGF-PLGA and BMSC (anti-miR-29) + bFGF from the LPP test displayed a very similar pattern.

[$] P < 0.05 vs PFD, BMSC (control) + bFGF, BMSC (control) + bFGF-PLGA and BMSC (anti-miR-29) + bFGF Results from the LPP test displayed a very similar pattern.

[$$] P < 0.01 vs PFD, BMSC (control) + bFGF, BMSC (control) + bFGF-PLGA and BMSC (anti-miR-29) + bFGF Taken together, regardless of bFGF source, transplant of control BMSCs did not exhibit any alleviating effect on the PFD rats.

[$$] P < 0.01 vs PFD, BMSC (control) + bFGF, BMSC (control) + bFGF-PLGA and BMSC (anti-miR-29) + bFGF Taken together, regardless of bFGF source, transplant of control BMSCs did not exhibit any alleviating effect on the PFD rats.

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Interestingly, miR-29b was drastically down-regulated by GH (21-fold, p = 0.008) (Figure 2b), GH-treatment did not affect the levels of 5S rRNA level (Figure 2a), suggesting a specific inhibitory effect of GH on miR-451 and miR-29b and putative roles for these miRNAs in the regulation of GH -dependent gene products.

Comparing these lists of putative miRNA targets for miR29b or miR-122a towards transcript profiles generated in our lab comparing gene expression in male and female rat livers, no transcripts were in common.

Since the main mechanism whereby miRNAs affect protein translatability is to inactivate/degrade the target mRNA, a lower level of miR-29b in response to GH might increase the amount of active/complete INSIG1 mRNA.

Among the miRNAs subjected to qRT-PCR, miR-29b was shown to be female-predominant and down-regulated in males given GH treatment.

Among the sex-different miRNAs listed in the table, only miR-21, miR-29b and miR-122a have been shown to be expressed in liver before [4, 6, 17- 22], implying that the other five might be "new" hepatic miRNAs.

Increased levels of e. g. miR-29b and miR-122a during starvation might reduce the translatability of key proteins for anabolic processes within the liver.

We conclude that mild starvation up-regulated the levels of miR-451, miR-122a and miR-29b significantly in both sexes, when compared to the postabsorptive state.

The female-predominant expression of miR-29b and miR-122a observed in this study might be of importance for metabolic control, since these miRNAs were shown to be increased during mild starvation, a condition when the liver switches from anabolic to catabolic pathways.

Combining in silico and in vivo studies, He et al identified a potential miR-29 target, insulin -induced gene 1 (INSIG1, or growth response protein CL-6) [17].

We conclude that hepatic miRNA levels depend on the hormonal and nutritional status of the animal and show that miR-29b is a female-predominant and GH-regulated miRNA in rat liver.

Among eight putative sex-different miRNAs, at least one female-predominant miRNA (miR-29b) could be confirmed using qRT-PCR.

Selected miRNAs (miR-122a, miR-29b and miR-451), and 5S rRNA as reference gene, were further examined using the miRCURY™LNA Real-time PCR kit (Exiqon, Danmark), according to the manufacturer's protocol.

Similar results were obtained for miR-29b, with increased levels in both males (8.5-fold, p < 0.001) and females (3-fold, p = 0.04) (Figure 3b).

Obviously, miR-29b is not a transcript that is female-predominant due to the female-specific pattern of GH.

Among the eight putative sex-different miRNAs, only one female-predominant miRNA (miR-29b) was confirmed using quantitative real-time PCR.

Furthermore, 1 week of continuous GH-treatment in male rats reduced the levels of miR-451 and miR-29b, whereas mild starvation (12 hours) raised the levels of miR-451, miR-122a and miR-29b in both sexes.

The biggest effects were obtained on miR-29b with GH-treatment.

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Furthermore, a recent study shows that upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury [25].

Note that miRNA expression levels of miR-29b, -324-3p and -347 were suppressed in earlier, but not later days (10-day) of hypoxia.

Although cultured neurons and astrocytes exposed to oxygen-glucose deprivation (OGD) exhibit diverse miRNA profiles, both cell types upregulate miR-29b in response to hypoxia [23].

Indeed, there is evidence showing that a neuroprotectant, IGF-1, downregulates miR-29b [23].

In addition, DOR activation accelerates the resolution of hypoxia -induced changes in miR-29b, -347 and -324-3p expression in the cortex.

Relative miRNA expression levels of miR-29b, -324-3p and -347 in the cortex following 1, 5 or 10 days of hypoxia or UFP-512 treatment.

Hypoxia alone reduced the expression of miR-29b by 40% after one day and 60% after 5 days.

However, miR-29b expression level almost returned to basal level after 10-day hypoxia.

A loss of miR-29b could increase calpain protein levels, inhibiting HIF1α and exacerbating the hypoxia-triggered flow reversal of the Na [+]/Ca [2+] exchanger [24].

If this is the case, the recovery of miR-29b after prolonger hypoxia for 10 days could be an adaptive response; however, physiological/pathophysiological implications of miR-29b may be more complex than we can presently imagine.

In the hypoxic animals, UFP-512 tends to decrease the level of miR-29b, especially at the time point of 10 days, though the change was not statistically significant (Figure 2A).

Similar to miR-29b, hypoxia and UFP-512 reduced miR-324-3p signals to barely 30% of the control (p<0.001).

21

Addition of UFP-512 suppresses these miRNAs and enhances the expression of a subset of mRNAs targeted by miR-21 and miR-29b.

On the other hand, DOR activation to prolong miR-29b suppression could lengthen the expression of HIF3a as well as its transcriptional regulators MAFG and MAFB.

The miR-29 family directly target at least 16 extracellular matrix genes and are relevant to renal and cardiovascular injury [29]– [31].

The downregulation of miR-21 and miR-29b produced by either hypoxia or DOR activation in 1-day group appeared to be reversed by day 5 unless the two treatments (hypoxia plus DOR activation) were given simultaneously.

Recent research suggests that miR-29 inhibits cell proliferation and induces cell cycle arrest [32], modulates oxidative injury [33] and promote apoptosis through a mitochondrial pathway that involves Mcl-1 and Bcl-2 [28].

The presence of UFP-512 alone suppressed miR-29b levels following 1 day of hypoxia exposure (Figure 8).

Several miRNAs (e. g., miR-29) that showed a dynamic change with hypoxic durations in this work regulate the process of oxidative stress and inflammation and are involved in regulation of apoptotic and survival signal pathways,.

Relative miRNA expression levels of miR-21 and miR-29b in the kidney following either 1, 5, or 10 days of hypoxia as determined by quantitative RT-PCR.

Either hypoxia or UFP-512 alone had no effect of miR-29b at 5 days, but the combination significantly stunted miRNA levels similar to miR-21.

Furthermore, hypoxic exposure for an even longer duration, i. e., 10 days, significantly increased both miR-29a and miR-29b levels that can explain induction of renal injury under prolonged hypoxia.

After 10 days, DOR stimulation significantly depressed miR-29b levels only in the presence of hypoxia.

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HL-1 atrial cardiomyocytes transfected with miR-29 and miR-200 (Fig 8) significantly down-regulate Cacna1c, Hnc4 and Ryr2 expression, while Camk2a was significantly decreased with miR-200 but not miR-29 (Fig 8).

miR-29 over -expression in HL1 atrial cardiomyocyte deregulate Cacna1c, Hnc4 and Ryr2, influencing therefore both the calcium handling and pacemaker activity, whereas miR-200 regulated Cacna1c, Ryr2 and Camk2a, in addition to Scn5a as previously reported [64], impacting therefore also in calcium handling.

Observe that miR-29 and miR-200 over -expression leads to significant decreased of Cacna1c, Hcn4, Ryr2 and Camk2a (except for miR-29a) expression.

We provide herein evidences that miR-29 and miR-200 over -expression also contributes to ion channel expression remo deling.

Thus these data demonstrate that miR-29 and miR-200 impaired expression also contributes to develop pro-arrhythmogenic substrates.

We have previously demonstrated that Pitx2 modulates expression of miR-29 and miR-200, among other microRNAs [16] and furthermore we have demonstrated in this study that modulation of distinct ion channel is greatly influenced by H [2]0 [2] administration while microRNA signature is mostly dependent on Pitx2c but not H [2]0 [2] administration.

Whereas it is wi dely documented that redox signaling can compromise ion channel functioning and calcium homeostasis in cardiomyocytes [67], in our system we observed no influence of H [2]O [2] administration on the regulatory impact of Pitx2 in distinct ion channels such as Scn5a, Kcnj2 and Cacna1c as well as multiple Pitx2-regulated microRNAs such as miR-1, miR-26, miR-29 and miR-200, in which redox impairment impact is less documented [68].

Importantly, miR-29 and miR-200 are not significantly impaired in SHR atrial chambers, suggesting that Wnt-microRNA might be a pivotal candidate establishing fundamental differences between HTD and HTN in atrial arrhythmogenesis susceptibility.

Modulation of miR-29 and miR-200 alters cardiac action potential determinants.

23

MicroRNA-29b directly binds to exon 3 coding sequence for TGF- β1 to promote its mRNA degradation and inhibit its translation (Zhang et al. 2014).

MicroRNA-29 overexpression and knockdown studies in cardiac cells and other cell types have reported its inhibitory action on the TGF- β1 pathway (Cushing et al. 2011; Luna et al. 2011; Roderburg et al. 2011; Zhang et al. 2014).

Angiotensin II also has been reported to regulate expression of microRNA-29 via Smads.

In summary, as outlined in Fig. 10 our study provides evidence that elevated IS levels caused by renal damage secondary to MI has a direct effect of on the expression of and microRNA-29.

MicroRNA-21 and microRNA-29 are among the most abundantly expressed microRNAs in heart and are known to regulate fibrosis by their action on mRNA of extracellular matrix proteins and TGF- β1 (van Rooij et al. 2008; Liang et al. 2012).

MicroRNA-29b levels were negatively and significantly correlated with its target genes collagen 1A1 (r [2] = 0.27; P = 0.005, Fig. 3D) and fibronectin-1 (r [2] = 0.17; P = 0.03, Fig. 3E).

Chronic angiotensin II infusion in wild-type mice resulted in significant reduction in microRNA-29 while in Smad3 knockout mice this reduction was prevented (Zhang et al. 2014).

Also, an important question, whether the uremic toxin IS promotes myocardial fibrosis by altering cardiac TGF β1-microRNA-21 and/or TGF β1-microRNA-29b signaling remains unexplored.

MicroRNA-29b mRNA expression in noninfarct myocardium was reduced by 50% in the MI+Veh group compared to sham animals (P < 0.01, Fig. 2B).

microRNA-29b.

MicroRNA-29b mRNA expression level in MI+AST-120 was significantly higher compared to the MI+Veh group (P < 0.001, Fig. 2B).

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These selected miRNAs included the seven most strongly upregulated miRNAs (miR-330, miR-338, miR-223, miR-20a, miR-181a, miR-592, miR-212) in the Ago2 IP at 30 min, the only downregulated miRNA (miR-29b) in the Ago2 IP at 30 min, and the three most strongly upregulated miRNAs (miR-219, miR-384, let-7f) in the Ago2 IP at 120 min post-HFS (significant by t-test with Dunn–Bonferroni correction and 1-Way ANOVA with LSD test).

In Ago2 IP samples, 14 miRNAs were upregulated at 30 min post-HFS, and only miR-29b was downregulated (Figure 2D).

Target gene list sizes for miRNAs with activity -dependent association with Ago2 for the 8 enhanced miRNAs were 97 (miR-20a), 156 (miR-219), 58 (miR-223), 114 (miR-29b), 30 (miR-330), 91 (miR-34a), 156 (miR-384), and 53 (miR-592) and for the 5 depleted miRNAs were 52 (let-7f), 55 (miR-338), 47 (miR-212), 255 (miR-19a), 32 (miR-326).

Five miRNAs (miR-384, miR-29b, miR-219, miR-592, and miR-20a) exhibited 2 to 5-fold greater increases in expression in the Ago2 immunoprecipitate than in the input samples, relative to contralateral control values (Figures 3C,D).

When comparing miRNA Ago2/input expression ratios, eight miRNAs (miR-384, miR-29b, miR-219, miR-592, miR-20a, miR-330 miR-223, and miR-34a) exhibited increases relative to the contralateral dentate gyrus, whereas five miRNAs (miR-let7f, miR-338, miR-212, miR-19a, and miR-326) showed decreases in this ratio.

Seven miRNAs (miR-384, miR-29b, miR-219, miR-592, miR-20a, miR-330, and miR-223) showed enhanced, NMDAR -dependent association with Ago2.

25

Among their shared miRNAs, previous research showed that miR-29 is significantly down-regulated and expressed in mesenchymal cells in the lungs of bleomycin -treated mouse [43].

We used TargetScan and miRanda database queries to obtain miRNAs, which had higher targeting combined with N4bp2, namely, miR-200, miR-429, miR-29 and miR-30.

miRNA-target prediction showed that MRAK088388 and N4bp2 had the same MRE for miR-29b-3p, whereas MRAK081523 and Plxna4 had the same MRE for let-7. To identify the ceRNA interaction between MRAK088388 and N4bp2, as well as between MRAK081523 and Plxna4, we detected whether they are co-expressed in lung tissues by using qRT-PCR.

By contrast, the levels of their shared miRNAs, miR-29b-3p and let-7i-5p, significantly decreased and was statistically correlated with overexpression lncRNAs respectively (Fig. 2).

Our qRT-PCR results show that the expression of miR-29 and MRAK088388 was highly correlated in lung tissue.

Based on these results and preliminary analysis, MRAK088388 possibly regulated lung myofibroblast growth and subsequent collagen deposition by sponging miR-29, which could bind to N4bp2.

26

It was recently demonstrated that miR-29b was dramatically down-regulated in the region of the fibrotic scar after MI and collagen expression in the heart was modestly increased in response to miR-29b inhibition [31].

The result demonstrated that miR-1 stressed upon regulation of myocyte growth, yet miR-29b and miR-98 put their regulatory emphases upon fibrosis and inflammation, respectively.

The modules regulated by miR-1, miR-29b and miR-98 contained more proteins encoded by ischemia related genes (Figure 3).

Further finding revealed that miR-1 focused on regulation of myocyte growth, yet miR-29b and miR-98 stressed on fibrosis and inflammation, respectively.

A-C. Largest functional modules regulated by miR-1, miR-29b and miR-98, respectively.

They were miR-1 emphasizing on cell growth regulation, miR-29b stressing on fibrosis and miR-98 focusing on inflammation.

Our network analysis identified that, among these miRNAs, the prime players in MI were miR-1, miR-29b and miR-98.

In contrast, we found an elevation in miR-29b.

27

Among the 14 miRNA upregulated in MSC-NCs, rno-miR-29a and rno-miR-29b were predicted to target REST analyzed by TargetScan (http://www.

Considering that miR-29a and miR-29b have overlapped predicted target genes and the expression change of miR-29a is more outstanding, we chose miR-29a for further studies.

Among those differently expressed microRNAs, miR-291a-5p, mir-294, miR-29a, and miR-29b were further detected by qRT-PCR, and the results were consistent with the microRNA array analysis (Fig. 2C).

0097684.g003 Figure 3. (A) miR-29a and miR-29b are predicted to target REST upon the neuronal differentiation of MSCs.

The human miR-29 family of microRNAs has three mature members, miR-29a, miR-29b, and miR-29c.

28

More direct evidence comes from a study using a mouse mo del of peritoneal dialysis, in which microRNA-29 (miR-29) was identified to be an inhibitor of peritoneal fibrosis through suppression of TGF-β signaling pathway [29].

The role of miR-29 family members in various fibrotic diseases such as renal and liver fibrosis has been reported, and the results indicate that they function through modulation of collagen related gene expression and formation of extracellular matrix [51, 52].

A recent study identified miR-29b, a downstream inhibitor of TGF-β signaling, as a negative regulator of mouse peritoneal fibrosis [29].

The change in expression of miR-29b between control and hypertonic dialysate groups was examined in our microarray analysis.

Presence of miR-29b has also been detected specifically in human peritoneal fluid [27].

miR-29b is a member of the miR-29 family which shares the same seed sequence.

29

The decrease of miR-29b expression can increase apoptosis markers while disinhibiting BACE1 expression to contribute to the production of Aβ in AD (Hébert et al., 2008; Kole et al., 2011; Delay et al., 2012), suggesting that altered miRNA expression may affect the pathogenesis of AD through a variety of mechanisms.

It has been shown that miR-29, miR-15 and miR-107 are upregulated; while miR-124, miR-34 and miR-153 are downregulated in patients with AD (Delay et al., 2012; Lau et al., 2013).

miR-29b is activated during neuronal maturation and targets BH3-only genes to restrict apoptosis.

30

MiRNAs exhibiting old-age expression included miR-21, miR-146a (Figure  6A,C), and members of the miR-29 family (miRa/b/c), which exhibit low expression at young age with increasing expression at older ages (78 and 104 weeks) (Figure  5J-L).

MiR-29b has also been shown to negatively regulate DNA methylation via its direct targeting of DNMT3A and DNMT3B, which are involved in de novo DNA methylation [46].

The miRNAs showing the highest expression within this group were three members of the miR-29 family (miR-29b, miR-29a, and miR-29c).

Furthermore, in diabetic conditions, expression levels of miR-195 and miR-29 have been shown to be elevated and reduced, respectively, in podocytes where they play a role in apoptosis and fibrosis [4].

Old age -associated miRNAs showed enrichment in pathways related to endocrine system disorders (miR-129-1, miR-375, miR-223, miR-664, miR-29b, miR-34a), cancer (miR-223, miR-29b, miR-375, miR-96), and cellular movement/invasion of cells (miR-29b, miR-29c, miR-7a, miR-96, miR-34a, miR-375).

MiR-29 family repression of DNA methylation activities in older animals would suggest multiple levels of epigenetic regulation.

31

As extracellular or cytoskeletal molecules are thought to be important in maintaining normal development, and their abnormal expression may cause pathogenesis, we chose Tm1α and 2β molecules, predicted targets of the miR29 family and used Western blot analysis to examine their protein expression in cell extracts isolated from LECs transfected with miR29a or 29c inhibitors (Fig. 4).

let7b, let7c, miR29b, miR29c and miR204 showed significant, gradual up-regulation (>2 times) after birth and during progression of lens development (ED16<4W<14W).

The increased expression of these molecules was inversely related to miR29 in LECs of SCRs with cataract.

32

We also identified differential expression of a number of miRNAs which target histone modification enzymes; miR-145 suppresses histone deacetylase 2 (HDAC2) [51], miR-129 is predicted to target HDAC2 mRNA and miR-29 targets HDAC4 mRNA [52].

In addition, 6 miRNA downregulated in our analyses (miR-26a, 29a/b/c, 222, and 383, Additional file 4: Table S1) are predicted to bind 3′UTR of Dnmt3B, with miR-222, miR-383 and miR-29b have demonstrated to directly affect Dnmt3B expression [49, 50].

33

Therefore, increased expression of miR-29 and miR-24 and reduced expression of miR-34, miR-130 and miR-378 may be responsible for the beneficial effects exerted by MSC-Exo.

Previous studies have shown that enhanced expression of miR-29 prevented kidney fibrosis by reducing the expression of collagen [32].

The expression of miR-29 and miR-24, which positively regulate cardiac functions, was relatively high (Figure 3(a)).

Our results showed high expression of miR-29 and miR-24 in both MSC-Exo and MSCs.

34

Remarkably, the main direction of changes of these genes (decrease in mRNA expression during maturation) is in agreement with the developmental increase of miR-29 expression under the assumption that miRNAs negatively regulate gene expression.

Notably, previously published studies provide some information about possible interactions between developmentally regulated miRNAs and genes, for example, regarding the miR-29 family.

One of the most strongly altered miRNA during postnatal development is the miRNA-29 family (miRNA-29a, -29b and -29c), that is associated with different signaling cascades.

Therefore, the finding of an established interaction between miR-29 and structural genes reported in the previous literature and our observation of differently expressed miR-29 and mRNA for collagen and elastin strongly suggest a possible interaction between these miRNAs and the regulated structural genes in the vessel investigated in our study.

35

S1 Fig The expression of MiR-29 was determined by qRT-PCR in the EDL muscle from 35 weeks old normal littermates (NL) and chronic kidney disease (CKD) rats.

The expression of MiR-29 was determined by qRT-PCR in the EDL muscle from 35 weeks old normal littermates (NL) and chronic kidney disease (CKD) rats.

We demonstrated in CKD skeletal muscle tissue there is higher stem cell activation (decreased Pax-7, increase MyoD) and differentiation (myogenin) with the lower expression of a pro-myogenic factor, miR-29.

In CKD there is lower expression of miR-29b compared to normal.

The expression of MiR-29b, a pro-myogenic and anti-fibrotic factor important during skeletal muscle cell differentiation [21], was significantly lower in CKD (0.96±0.11 vs.

Target-specific PCR primers (Pax-7, MyoD, Myostatin, Myogenin, Atrogin 1, MuRF-1, miR-29b, Activin 2b, SOD-1, and SOD-2) were obtained from Applied Biosystems.

36

Upregulation or downregulation of miRNAs previously implicated in aGvHD pathogenesis such as the miR-17-92 cluster, miR-29a, miR-29b, miR-146a, or miR-155 did not reach significance in the NanoString analysis.

Particular miRNAs associated with aGvHD include miRNAs that enhance T-cell activation, such as miR-155 (4, 10), miR-142 (11), miR-29a, miR-29b, and the miR-17-92 cluster (12), and miRNAs that repress T-cell activation, such as mir-146a (13), which is also upregulated in T regulatory cells (Tregs) (14).

We found clear upregulation of miR-146a and miR-155, but not of miR-29a, miR-29b, miR-19b, or miR-20a in skin (Figure 4A and data not shown).

37

The analysis based on their fold changes showed a significant (P < 0.05) upregulation of miR-200c-3p (predicted to regulate IL-13 and VEGF-alpha), miR203a-3p (predicted to regulate IL-24 and PRKC α), miR29-3p (predicted to regulate TNFRS1A), and miR-21-5p (predicted to regulate NFk-B activity), in ocular tissues of LPS+RvD1 -treated rats compared to the vehicle+LPS group (Figure 4).

These were miR-21-5p that is predicted to regulate NFk-B activity; miR-200c-3p that is predicted to negatively regulate IL-13, LEPR, NTF3, PRKC α, RIPK2, and VEGFA indicating decreased of proinflammatory cytokines [29]; miR-203a-3p predicted to regulate IL-24 and PRKC α; miR-29b-3p predicted to negatively regulate HDAC4, IL-1RAP, Lif, PDGF α, PDGFc, VEGFA, and TNFRSF1.

Interestingly, upregulation of miR29-3p and miR-21-5p induced by RvD1, significant already at the lowest dose of 10 ng/kg, was concomitant with the decrease of TNF- α and NF- κB levels in the ocular tissue (Figures 5 and 6).

38

From P4 to P28, miRNAs displayed very similar expression between both progeny: 0–22 (<5%) miRNAs displayed differential expression and 0–7 of them (<2%) had expression differences higher than 3. Ratios of miR-7a-5p, miR-24-3p, miR-29-3p, miR-137-3p or miR-1843-5p expressions relatively to miR-124-3p expression calculated from RT-qPCR at P28 data matched those calculated from HTS data (Fig. 3B).

As the U6 snRNA was excluded from small RNA fractions and could not been used as an internal reference to quantify miRNA expressions, we quantified the expression of miR-7a-5p, miR-24-3p, miR-29-3p, miR-137-3p and miR-1843-5p relatively to that of miR-124-3p, at P4, P8, P14.4 and P21.4 relatively to P28, from RT-qPCR amplification or sequencing data (Fig. 3A).

Expression of miR-7a-5p, miR-24-3p, miR-29-3p, miR-137-3p and miR-1843-5p were quantified relatively to those of miR-124-3p, and relatively to those of stage P28, by using the ΔΔCt method 25 and experimentally ascertained amplification efficiencies.

39

Finding out biological function of miRNAs, proved its expression and regulation mechanism eventually, will have profound significance for better understanding of IBS-D. In a word, our findings may lead to new therapeutic targets [34] for the treatment of IBS-D with increased intestinal permeability via miR-29 regulation.

Study displayed the up-regulated miRNA-29 resulted in the increased intestinal permeability [24].

Zhou [24] found that miR-29 targets on nuclear factor-κB-repressing factor and Claudin 1 to increase intestinal permeability.

MiRNA-29a is a member of miRNA-29 family.

40

Studies have shown that AET increases microRNA-29 expression in the heart and consequently decreases collagen expression and protein levels [24, 25].

To confirm the involvement of obesity-regulated microRNAs in pathological CH, we analyzed the cardiac microRNA-29 family (microRNA-29a, microRNA-29b and microRNA-29c), whose expression affects collagen content.

The relative expression of COLIAI, COLIIIAI, ANF, α-MHC, α-actin skeletal, β-MHC, microRNA-1, microRNA-29a, microRNA-29b, and microRNA-29c was analyzed using real-time polymerase chain reactions (real-time PCR) as described previously [24].

The microRNA-29 family has been described to negatively regulate collagen content and to be highly responsive to AET [22, 24, 25].

41

Klotho deficiency and normal ageing may be associated with upregulation of miR-29a and miR-29b [34], and miR-29a is associated with inflammation as well [33].

Members of the miR-29 family suppress the protein phosphatase PPM1D, increasing apoptosis [30], and may function as markers of senescence because they can reduce the levels of type IV collagen, potentially weakening the basal membrane in senescent tissues [28].

Also on D2, the expression of miR-29b and miR-335 (Fig.   4g, h) did not differ significantly among the IRI, IRI + huMSC and control groups: 7.7 ± 6.8, 5.1 ± 4.3 and 2.2 ± 2.0, respectively, for miR-29b; and 0.5 ± 0.0, 0.2 ± 0.1 and 1.1 ± 0.4, respectively, for miR-335.

g Bar graphs showing renal miR-29b expression.

The miRs studied were miR-29a, miR-29b, miR-335 and miR-34a (Applied Biosystems; Thermo Fisher Scientific).

42

In the present study the mRNA microarray experiment showed that many of the targets of miR-29 were indeed significantly down-regulated, both in WKY and SHR.

MiR-29 has previously been related to aging and dilation of the aorta, associated with down-regulation of extracellular matrix molecules [18].

Thus, maturation in both WKY and SHR vessels was associated with a prominent increase in expression of the miR-29 family.

This is confirmed by a recent study where miR-29b was related to a reduction in extracellular matrix proteins as shown by a proteomic approach [19].

43

On day 7, miR-31, miR-214, miR-199a-5p, and miR-199a-3p were up-regulated, whereas miR-181c, miR-29b, miR-26b, miR-181d, mir-126, mir-499-5p, and miR-1 were down-regulated.

Some of the deregulated miRNAs (miR-181, miR-26, miR-1, mir-29, miR-214, miR-126, and miR-499) are reported to be related to hypoxia, cell development, and cell growth [1, 5, 7, 25].

Recently, some miRNAs for example miR-21, miR-1, miR-216[10], and miR-29 family[11], were reported to be deregulated in myocardial infarction.

44

In the mo del group, 17 miRNAs were downregulated, including miR-1, miR-133, miR-29, miR-126, miR-212, miR-499, miR-322, miR-378, and miR-30 family members, whereas the other 18 miRNAs were upregulated, including miR-21, miR-195, miR-155, miR-320, miR-125, miR-199, miR-214, miR-324, and miR-140 family members.

Among these differentially expressed miRNAs, miR-1, miR-133, miR-29, miR-126, miR-499, miR-30, miR-21, miR-195, miR-155, miR-199, miR-214, and miR-140 have been reported to be related to MI [25– 36], while the other miRNAs have not been reported directly in MI.

45

Thyrotropin Regulates Thyroid Cell Proliferation by Up-Regulating miR-23b and miR-29b that Target SMAD3.

In thyroid cells, miR-23b and miR-29b can promote cell growth by targeting Smad3 [27].

For example, miR-105, miR-125b and miR-140 are involved in the inflammatory phase; miR-15a, miR-15b and miR-16 participate in the granulation phase; and miR-29 and miR-192 function in the remo deling phase [10].

46

The over -expression of miR-29 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose uptake [37].

The miR-29 group of microRNAs was found to be up-regulated in muscle and fat tissues of Goto–Kakizaki rats, a non-obese rat mo del of diabetes mellitus (T2DM).

This small microRNA miR-29b is serving also as an internal control because this regulatory molecule turned out to be not significant.

47

miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family and that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family.

The inconsistency between Zhang’s report and our study suggested that miRNA patterns in the organ of Corti change with aging and that miRNAs such as miR-183 and miR29 play different roles in the development of organ of Corti in newborn, younger and older animals.

48

Furthermore, miR-29b has a role in the regulation of GRN expression levels in a stable cell line (hPGRN-3T3) expressing full-length human GRN cDNA (including the 3′UTR; Jiao et al., 2010).

MicroRNA-29b regulates the expression level of human progranulin, a secreted glycoprotein implicated in frontotemporal dementia.

49

MiR-29 family members are often down-regulated in cancer and forced expression of miR-29a is reported to reduce proliferation and invasiveness presumably via CDC42 and p85alpha dependent up-regulation of p53 [36], [37].

50

Among the miRNAs, miR-34a, miR-32, miR-376a, miR-384-3p, miR-29b and miR-142-3p were the highly overexpressed, with fold induction of 11.8, 24.2, 12.7, 14.4, 11.6 and 19.1, respectively.

We noted that miRNAs miR-34a, miR-18a, miR-19a, miR-32, miR-96, miR-142-3p miR-29b and miR-7b were significantly upregulated in the AOM rat fecal colonocytes compared to those obtained from the saline controls and the degree of induction was greater in the tumor bearing AOM rats compared to the tumor non-bearing AOM rats (Fig. 3B).

Out of the 12 miRNAs tested, four (miR-21, miR-18a, miR-29b, and miR-19a) were significantly different between AOM and Saline group (p<0.02).

In the 16 week colonic biopsies, we observed that while all miRNAs trended to increase (versus age-matched saline treated animals) although only 7 miRNAs (miR-34a, miR-21, miR-18, miR-376a, miR-19a, miR-9 and miR-29b) achieved statistical significance (fold inductions of 1.73, 2.72, 2.15, 2.26, 2.18, 1.53, and 1.71,respectively) (Table 2).

Based on these criterion miR-21 achieved distinguished predictive ability with a 0.914 AUC, miR-18a and miR-19a achieved excellent predictive ability with a 0.877 and 0.872 AUC respectively, miR-29b achieved an almost excellent predictive ability with a 0.789 AUC.

51

Of these miRNAs, rno-miR-129-1-3p, rno-miR-153-3p, rno-miR-29b-3p, rno-miR-29c-3p and rno-miR-451-5p were down-regulated, whereas rno-let-7a-1-3p, rno-miR-322-5p, rno-miR-3574 and rno-miR-628 were observed to be highly upregulated with p < 0.01 (Fig.   3).

9 miRNAs (rno-miR-129-1-3p, rno-miR-153-3p, rno-miR-29b-3p, rno-miR-29c-3p, rno-miR-451-5p, rno-let-7a-1-3p, rno-miR-322-5p, rno-miR-3574 and rno-miR-628) showed statistically significant change.

52

MicroRNA-29a and 29b-1 are processed from chromosome 7, and miR-29b-2 and 29c are transcribed from chromosome 1. Since forced overexpression of miR-29 markedly suppresses collagen 1 A1 mRNA and protein expression [16, 17], miR-29 plays an anti-fibrotic role in the liver and other organs [18].

The miR-29 group is composed of miR-29a, 29b-1, 29b-2, and 29c.

53

The let-7d 27 and miR-29 28 down-regulation, and the miR-21 up-regulation 29 contribute to PF.

54

For example, on a northern blot miR-29b was almost undetectable at the embryonic and P4 stages but appeared at P18 and was strongly expressed in the adult.

Examination of the temporal clusters revealed that probes with similar sequences showed correlated expression, as exemplified by miR-181a, miR-181b, miR-181c, smallRNA-12 (Figure 4a) and miR-29a, miR-29b and miR-29c (Figure 4b), respectively.

The probes used were: EAM119 (miR-29b), EAM125 (miR-138), EAM224 (miR-17-5p), EAM234 (miR-199a), EAM131 (miR-92), EAM109 (miR-7), EAM150 (miR-9) and EAM103 (miR-124a).

55

As was shown by the recent paper of Kalani et al. (2014), this process could be regulated by epigenetic mechanisms including expression of the specific miRNA29b.

Moreover, Hcy itself directly affects the BBB permeability and the miR29b regulated activity of MMP9 seems a novel epigenetic mechanism.

56

Importantly, the miR-29 family was exclusively downregulated in the frontal cortex, resulting in upregulation of methyltransferase DNMT3a [21, 23].

57

The results showed that 14 miRNAs (miR-30a-5p, miR-30e-5p, miR-425-5p, miR-142-3p, miR-191a-3p, miR-215, miR-29b-3p, miR-30b-5p, miR-26a-5p, miR-345-5p, miR-361-5p, miR-185-5p, miR-103-3p) were down-regulated but no miRNA was up-regulated among above three altered miRNAs from microarray in OVX serum by normalizing to miR-25-3p (Fig. 3b).

58

In an elegant study, Van Rooij [14] showed that downregulation of miRNA-29 increased collagen expression and fibrosis in the heart after MI.

Wang G, Kwan BC-H, Lai FM-M, Chow K-M, Li PK-T, Szeto C-C. Urinary miR-21, miR-29, and miR-93: novel biomarkers of fibrosis.

59

Since the expression of LOXL2 is also influenced by hypoxia,, and microRNAs (miR-26 and mIR-29), there are also other potential strategies for targeting LOXL2 expression or activity (Wong et al., 2014).

60

The most upregulated miRNAs were miR-146a-5p, miR-132-5p, miR-21-5p at 1.63, 1.61, and 1.56-fold of the control, respectively, while the most downregulated miRNAs were miR-29b-3p, miR-352, miR-30e-5p at 0.60, 0.70, and 0.72-fold of the control, respectively.

61

MicroRNA miR-29b is known to increase during neuronal maturation and to inhibit apoptosis in neurons, and miR-29a/b both affect dendritic spine morphology (Kole et al. 2011; Lippi et al. 2011).

The members of the miR-29 family showed the highest increase in expression level from younger to older animals (Fig.   1d; log fold-change (LFC) of up to 5.5 from P2/P9 to P23/P45).

Three of the top five most significant miRNAs that increased from early to late age were members of the miR-29 family.

62

For instance, miR-29b has been approved to induce global DNA hypomethylation and reactivate tumor suppressor gene P15 and ESR1 in acute myeloid leukemia by targeting DNMT3A and DNMT3B directly and DNMT1 indirectly (18).

63

Northern blot analysis confirmed the up-regulation of all three miR-29 paralogs in muscle, adipose tissue and liver.

He et al. (2007) [19] profiled miRNA expression in skeletal muscle in the Goto-Kakizaki (GK) rat, a well-characterized mo del of T2D, and compared it to normal Wistar rats; miR-29 paralogs were found to be over-expressed in the GK rat.

64

Downregulation of mir-29 (mir-29a and mir-29c) by antisense inhibitor also protected H9c2 cardiomyocytes from simulated IR injury.

65

By contrast, miR-29b appears to act as a beneficial factor that protects against liver fibrosis by suppressing the activation of HSCs [11].

A previous study suggested that miR-29 regulates liver fibrosis and together with TGF- β and nuclear factor- κB forms part of a signaling nexus in HSCs [40].

It has also been shown that hepatic levels of miR-29 are significantly increased in mice with CCl4 induced liver damage and also in the livers of patients with advanced fibrosis.

66

In diabetic nephropathy, the miRNA-29 family protects the kidney from fibrotic damage, and the DPP-4 inhibitor linagliptin has been shown to inhibit TGF-β -induced endothelial to mesenchymal transition (EndMT) by restoring the miRNA-29s’ levels [64].

Nevertheless, the miR-29 family might serve as a candidate for monitoring of treatment effects.

67

For example, miR-29 expression is downregulated in human and murine liver fibrosis, which is mediated by TGF-β and other inflammatory signals [16].

68

Moreover, the downregulation of miR-29, which is a modulator of ECM homeostasis [106], may induce the overexpression of key pro-regenerative matrix molecules, such as laminin, collagen, and fibronectin.

69

Moreover, miR-29 has target specific sites on BACE1 mRNA and its down-regulation increases AD progression [65].

70

Another gene family that we evidenced prevalently expressed in the WBCs is mir-29 (mir-29a, mir-29b, mir-29c; p = 2.19 E-4).

We found that miRNAs with higher expression in WBCs includes different miRNA families: mir-15, mir-17, mir-181, mir-23, mir-27 and mir-29 families.

71

Uematsu et al. [32] reported that regulating the expression of SRY-box 4 (SOX4) and lymphoid enhancer -binding factor 1 (LEF1) by miR-29b-1-5p and miR-449a-5p are important in the development of Th2 bias in methimazole -induced liver injury.

72

For example, miR-101 and miR-29b both target Mcl-1 to prevent apoptosis 6, 7, while miR-499a impairs myocyte survival by repressing the expression of histone deacetylases [8].

73

On the other hand, the levels of expression in colostrum whey and mature milk whey of miR-29b, and miR-223 are different from those of bovines [4].

However, in rat, miR-29b was significantly higher in d 9 milk whey than in d 2 colostrum whey, and there was no difference in miR-223 in colostrum whey and in mature milk whey.

That is to say, in comparison between colostrum and mature milk whey by qPCR in bovine, there was no significantly difference in miR-29b between colostrum and mature milk, and miR-223 was significantly higher in colostrum whey than in mature milk whey.

74

Comparatively, the miR-29 family showed consistent regional expression [25] while the miR-200 family were differentially expressed.

75

Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis.

Expression of MicroRNA-29 and collagen in cardiac muscle after swimming training in myocardial-infarcted rats.

76

Du Y Upregulation of a disintegrin and metalloproteinase with thrombospondin motifs-7 by miR-29 repression mediates vascular smooth muscle calcificationArterioscler.

77

A recent study reiterated the role of miR-29 in neuronal survival by knocking down miR-29 in the mouse brain [73].

miR-29 knockdown resulted in massive neuronal death in the hippocampus and cerebellum.

78

Dramatical down-regulation of miR-29 was observed in the region of the fibrotic scar after MI [31].

79

Furthermore, among these miRNAs, many were found to be involved in angiogenesis, including several with a regulatory function in cell proliferation, the expression of growth factors and extracellular proteolysis, such as miR-497, miR-98, miR-181a, miR-145, miR-29b and miR-27a 24– 29.

80

Maegdefessel L. Azuma J. Toh R. Merk D. R. Deng A. Chin J. T. Raaz U. Schoelmerich A. M. Raiesdana A. Leeper N. J. Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development J. Clin.

81

Recent studies have identified that many miRs [11], such as miR-1, miR-21, miR-29, miR-31, miR-143/145, and miR-221/222, play important roles in neointimal hyperplasia by regulating the functions of VSMCs.

A number of miRs, such as, miR-1, miR-21, miR-29, miR-31, miR-143/145 and miR-221/222, were verified to be involved in neointimal hyperplasia by regulating the functions of VSMCs [11].

82

For example, miR-206 and miR-29b are known to be significantly upregulated in the context of diabetes [27, 28] and such increase was confirmed in our human cohort.

83

Gomes PR Long-term disruption of maternal glucose homeostasis induced by prenatal glucocorticoid treatment correlates with miR-29 upregulationAm.

84

revealed that four miRNAs (miR-29b-3p, miR-145-5p, miR-24-2-5p, miR-665) were significantly regulated (P<0.05), three miRNAs (miR-21-3p, miR-466b-2-3p, miR-466d) tended to be significantly regulated (P<0.15), and one miRNA (miR-34a-5p) was not confirmed to be significantly regulated by qRT-PCR (Table 2 ).

85

Ventral combined with dorsal root avulsion resulted in a sustained upregulation of 10 miRNAs, including miR-19b-3p, miR-20b-5p, miR-21-5p, miR-27a-3p, miR-29b-3p, miR-106b-3p, miR-142-3p, miR-322-5p, miR-352, and let-7a-5p (Figure  2E).

86

Lovastatin upregulates microRNA-29b to reduce oxidative stress in rats with multiple cardiovascular risk factors.

87

For instance, some miRNAs, such as miR-29, miR-21 and miR-221, has been reported to regulate tumor cell growth, apoptosis, migration and invasion by targeting proteins involved in those cellular pathways [7- 9].

88

He et al (23) reported an elevated expression of the miR-29 gene family in skeletal muscle, liver and adipose tissues of diabetic GK rats.

89

Additionally, Zhang et al. [22] have demonstrated miR-29b and miR-142-5p that was changed significantly in human serum during aging process, and we have confirmed that these two miRNAs also showed same expression pattern in young and aged rat serum (Supplementary Figure 2).

90

We identified a group of mRNA and microRNA previously associated with amyloid-ß induced toxicity (e. g. Frp2 and Ppif), or implicated in Alzheimer’s disease processes, (miR-29 and miR-9) [35- 47], (Table  4).

91

For example, it has been reported that miR-221 [15], miR-199a/b [16][17], miR-27b [18], miR-195 [11] and miR-34a/b/c [19] positively regulate cardiac hypertrophy, while miR-378 [9], miR-29 [20], miR-150 [11], miR-223 [21] and miR-1 [22] negatively regulate cardiac hypertrophy.

92

Melo S. T. F. Fernandes T. Baraúna V. G. Matos K. C. Santos A. A. Tucci P. J. F. Oliveira E. M. Expression of microRNA-29 and collagen in cardiac muscle after swimming training in myocardial-infarcted rats Cell.

93

Overexpression of miR-34a, miR-146a, miR199a-5p or miR-29 in MIN6 cells negatively impacts on beta cell function [6].

94

Putative interactions that were specifically found for NGCs include the anti-correlation between the miRNA rno-miR-29b and its potential target mRNAs, Sgk1, and Mgat4.

95

The expression of mir-29b and mir-133 in the heart has been confirmed by northern blot [19].

96

As an example, this situation has been demonstrated for miR-29 released from cancer tissue and targeting skeletal muscle cells, which triggers cytopathic effect and cachexia [120].

97

Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction.

98

Expression of miR-29b, -124a, -155 and -223, that were significantly increased in the atherosclerotic abdominal tissue was reduced in circulation.

99

As shown in Table 2, we found increased expression of liver specific miRNAs in transdifferentiated hepatocytes, including miR-122a, miR-21, miR-22, miR-182, miR-29 and miR-30.

100

Eisenrech et al. [17] demonstrated that miR-29b is involved in the regulation of apoptosis in podocytes through the regulation of podoplanin.