59 publications mentioning rno-mir-30e

Open access articles that are associated with the species Rattus norvegicus and mention the gene name mir-30e. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

CaMKIIδ has been reported to regulate VSM proliferation through signaling pathways that result in suppression of p53 and p53-targets including CDKN1 (p21), a cell cycle inhibitor 26. miR-30 family members have been reported to function as tumor suppressors by inhibiting cell proliferation, invasion and inducing apoptosis through different mechanisms, such as targeting BCL9 and repressing Wnt signaling 36.

Using well-established aortic VSM primary cell culture and vascular injury mo dels, we demonstrated; 1) an inverse correlation between CaMKIIδ protein and miR-30 family expression in VSM cells upon phenotype switching in vitro and in response to vascular injury in vivo; 2) regulation of CaMKIIδ expression and cell proliferation by miR-30 in cultured VSM with magnitude changes comparable to those observed in vivo following vascular injury; and 3) prevention of CaMKIIδ upregulation with inhibition VSM cell proliferation and neointima formation in vivo following ectopic miR-30 expression.

Since we demonstrated that miR-30 significantly inhibited VSM proliferation in vitro, in part by targeting CaMKIIδ expression, we tested effects of ectopic miR-30 expression on CaMKIIδ expression and vascular remo deling in vivo following rat carotid artery balloon injury.

Overexpression of miR-30 reduces CaMKIIδ expression by targeting CaMKIIδ mRNA 3′UTR and inhibits VSMCs proliferation.

In the present studies, miR-30 overexpression decreased CaMKIIδ expression and inhibited VSM proliferation in vitro, an effect that could be partially rescued by re -expression of CaMKIIδ to physiological levels.

Based on reciprocal expression dynamics between CaMKIIδ and miR-30 in VSM in vivo in response to vascular injury, we tested the hypothesis that CaMKIIδ is in fact an endogenous target of miR-30 and its expression is regulated by miR-30.

Because the synthetic phenotype cultured VSM cells expressed low levels of miR-30 family members, and effective miR-30 silencing would require siRNAs targeting all of the redundant family members, we relied on gain-of-function approaches to test if CaMKIIδ was, in fact, a direct target for miR-30.

Overexpression of miR-30 significantly inhibits CaMKIIδ protein expression in cultured VSMCs.

Because miRs typically have multiple mRNA targets we determined to what extent CaMKIIδ protein expression could rescue miR-30e induced inhibition of VSM proliferation.

CaMKIIδ [2] is involved in miR-30 mediated attenuation of VSM cell proliferationBecause miRs typically have multiple mRNA targets we determined to what extent CaMKIIδ protein expression could rescue miR-30e induced inhibition of VSM proliferation.

Based on these reports, the current studies, and one report that identified miR-30b as a regulator of CaMKIIδ expression in cardiomyocytes, it is reasonable to propose that dysregulation of a miR-30/CaMKIIδ axis in both VSM and heart might contribute to diverse cardiovascular diseases.

Additionally, when Shi et al. induced apoptosis using TGF-β in podocytes, miR-30 expression was observed to be down-regulated in a Smad2 -dependent manner 48.

Previous studies have suggested coordinate down-regulation of miR-30 family members in skeletal muscle as a function of de-differentiation or dystrophic disease 41, in cardiac muscle as a function of hypertrophy heart failure 42 or atrial fibrillation 43, in human thoracic aortic dissection 44, and in cardiac and VSM cells following ER stress 45 46.

Importantly, lentiviral transduction of miR-30 in vivo, immediately following injury, largely prevented subsequent CaMKIIδ upregulation and strongly inhibited VSM cell proliferation and neointimal remo deling.

MiR-30 inhibits CaMKIIδ expression by targeting CaMKIIδ 3′UTR in primarily cultured smooth muscle cells.

In primarily cultured rat VSM cells, CaMKIIδ [2] expression was inhibited approximately 50% after introduction of miR-30c or miR-30e mimics introduced by electroporation (Fig. 2b).

These experiments confirm CaMKIIδ as a target of miR-30 and mediator of miR-30 induced inhibition of cell proliferation.

In these experiments we titrated adenoviral transduction of a wild-type CaMKIIδ [2] construct into VSM overexpressing the miR-30e mimic such that CaMKIIδ expression after 48 and 72 hr was rescued to control levels (Fig. 4a; top panel).

miR-30c and miR-30e mimics significantly reduced CaMKIIδ protein expression by about 50% and inhibited activity of a luciferase-CaMKIIδ 3′-UTR construct by 30%.

Overexpression of CaMKIIδ partially rescues VSM proliferation inhibition by miR-30e.

MiR-30 family member expression is significantly decreased following balloon angioplasty injury of rat carotid arteries coincident with CaMKIIδ upregulation and neointimal vascular remo deling.

miR-30e transduction had no effects on VSM apoptosis (data not shown) but significantly inhibited PCNA expression after 72 hr, a marker of active cell proliferation (Fig. 3c).

Similarly, comparing miR-30 expression in differentiated VSM from intact aorta medial layers to synthetic phenotype cultured aortic medial VSM cells confirmed reduced expression of miR-30a-e in de-differentiated cells by 30–70%.

Partial rescue suggests there are other targets of miR-30 in addition to CaMKIIδ that could be involved in miR-30 induced inhibition of VSM cell proliferation.

Given the diversity of pathways impinging on cell cycle control, multiple signaling pathways may also be regulated in VSM by miR-30 and contribute to its net inhibitory effects on VSM proliferation.

Deletion of the putative miR-30 binding sequence in the CaMKIIδ 3′-UTR construct abrogated the effects of miR-30, supporting the hypothesis that CaMKIIδ is a direct target for miR-30 in VSM.

Neointima formation is a balance between VSMC proliferation and apotosis, and our data does not exclude the possibility that miR-30 regulates VSMC apoptosis in vivo, thus contributing to its inhibitory effect on the neointima formation.

How to cite this article: Liu, Y. F. et al. MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca [2+]/calmodulin -dependent protein kinase IIδ (CaMKIIδ).

Next, we employed a luciferase reporter assay to determine if miR-30 directly interacts with CaMKIIδ 3′UTR, and exert its inhibitory regulation.

miR-30c, the most abundant miR-30 family member in vivo, was introduced into the medial wall by lenti-viral transduction immediately after carotid injury in order to rescue injury -induced miR-30 downregulation.

We expect that miR-30, miR-145 and other miRs act collectively to regulate the VSM phenotype switch which involves changes in expression of hundreds of proteins.

Thus, it needs to be studied if miR-30 interferes with the activation or promotes differentiation of vascular progenitor cells, resulting in the inhibition of neointima hyperplasia.

Lenti-viral delivery of miR-30 to the injured carotid artery walls dramatically inhibits cell proliferation in the medial walls and neointima hyperplasia.

Collectively, in differentiated carotid arteries (Fig. 1) and aorta (Fig. 2) miR-30 family members are expressed at levels comparable to miR-145 which is considered a high abundance microRNA associated with and regulating the differentiated VSM phenotype 23.

Furthermore, overexpression of miR-30 impairs cultured VSM cell proliferation, and CaMKIIδ rescue in the presence of miR-30 partially, but significantly recovers VSM cell growth.

The expression of miR-30 family members is reduced in dedifferentiated vascular smooth muscle.

Collectively, miR-30 family members are expressed at a level comparable to miR-145.

All miR-30 members were expressed in uninjured carotid medial VSM with miR-30c being the most abundant (Fig. 1d).

Based on in silico analysis of the rat CaMKIIδ 3′UTR sequence (TARGETSCAN), a miR-30 family binding sequence is predicted ([+2036]UGUUUACA [+2043]) (Fig. 1c).

In summary, we have shown that elevated CaMKIIδ protein and attenuated miR-30 expression in dedifferentiated/synthetic vascular smooth muscle cells in vitro and in vivo.

To study if miR-30 binds to CaMKIIδ 3′UTR, full-length of CaMKIIδ 3′UTR (Accession: NM_012519.2, +1825 to +2300) and truncated CaMKIIδ 3′UTR (+1825 to +2035) without miR-30 binding site ([+2036]UGUUUACA [+2043]) were cloned into pMIR-REPORT™ miRNA Expression Reporter Vector (Invitrogen).

7 days post vascular injury, expression of all miR-30 family members was significantly reduced in medial wall VSM by 60–80%.

miR-30e over -expression at 72 hr was confirmed by qPCR (Fig. 3b).

As shown above, miR-30e overexpression slowed VSM cell growth by 72 hr (Fig. 4b).

Mechanisms underlying coordinate regulation of miR-30 family members are not known.

Interestingly, miR-30 family members are encoded on 3 different chromosomes in clustered pairs: miR30a and –c2 on human chromosome 6, miR-30b and-d on chromosome 8, and mir30e and –c1 on chromosome 1. A recent report indicates human coordinate regulation of several miR-30 family members by Mef2 in skeletal muscle 47.

Vascular injury induces reciprocal regulation of miR-30 family members and CaMKIIδ protein in rat carotid artery.

The expressions of miR-30 family members and miR-145 were compared between injured and uninjured carotid arteries and U6 expression was measured and used for normalization.

Moreover, it has been reported that miR-30 regulates epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) in various tumor cells 39 40.

MiR-30 inhibits vascular smooth muscle cell proliferation.

The significance of the latter experiment is two-fold; confirming miR-30 dependent regulation CaMKIIδ in vivo and suggesting the therapeutic potential for miR-30, at least in the context of restenosis.

In the present studies we focused on CaMKIIδ regulation by miR-30 family members which are predicted to bind to a species-conserved sequence in the CaMKIIδ 3′-UTR.

CaMKIIδ [2] is involved in miR-30 mediated attenuation of VSM cell proliferation.

Cultured VSM cells were eletroporated with scrambled miRNA or miR-30e mimic (0.2 pmol) and after 24 h, adeno-virus encoding CaMKIIδ2HA or GFP (2.5 MOI) was utilized to infect electroporated VSM cells for CaMKIIδ rescue.

These magnitude changes are comparable to those previously observed in A10 cells using miR-145 23 and the partial effects in both studies could be due redundant effects of the miR-30 and miR-145 species.

A full-length CaMKIIδ 3′UTR construct and truncated CaMKIIδ 3′UTR construct without the predicted miR-30 seed sequence ([+2036]UGUUUACA [+2043]) were cloned into a luciferase reporter vector (Fig. 2c).

Upon vascular injury or culture of the VSM cells, total miR-30 levels decrease by approximately 75%, similar in magnitude to the decrease in miR-145 and coincident with the switch to a VSM cell synthetic phenotype.

All experiments were carried out with VSM cells from passage 3 to 6. To transduce miR-30 into the common carotid artery wells in vivo, we produced lenti-virus encoding miR-30c using HIV -based pPACKH1 packaging system (System Biosciences, CA, USA).

2

To do this, we injected the neurone -targeted gene knock-down system (LV-mCMV/SYN-tTA + LV-Tretight-GFP-miR30-shRNA/Luc) with LVV to express Luc in astrocytes (LV-GfaABC [1]D-Luc) and, conversely, the astrocyte -targeted knock-down system (LV-mCMV/GfaABC [1]D-tTA+ LV-Tretight-GFP-miR30-shRNA/Luc) together with LVV for neuronal Luc expression (LV-SYN-Luc).

LTR, lentiviral long terminal repeats; Tretight, a modified tetracycline and Dox-responsive promoter derived from pTRE-tight (Clontech); GFP, green fluorescence protein; miR30-shRNA/Luc, miR30 -based shRNA targeting firefly Luc gene; miR30-shRNA/nNOS, miR30 -based shRNA targeting rat neuronal nitric oxide synthase gene; Luc, firefly Luc gene; GfaABC [1]D, a compact glial fibrillary acidic protein promoter (690 bp); SYN, human synapsin 1 promoter (470 bp); mCMV, minimal CMV core promoter (65 bp); GAL4BDp65, a chimeric transactivator consisting of a part of the transactivation domain of the murine NF-κBp65 protein fused to the DNA binding domain of GAL4 protein from yeast; WPRE, woodchuck hepatitis post-transcriptional regulatory element.

To this end, we constructed a binary Dox-controllable and cell-specific miR30 -based RNAi system to express shRNAs targeting a reporter gene for Luc and an endogenous gene for nNOS.

a: LV-mCMV/GfaABC [1]D-tTA controlled miR30-shRNA/Luc didn't knockdown Luc expression in neurones.

b: LV-mCMV/SYN-tTA controlled miR30-shRNA/Luc didn't knockdown Luc expression in glia.

These results demonstrate that bidirectional transcriptionally amplified SYN and GfaABC [1]D promoters provide a sufficient level of tTA to activate the Tretight promoter which then drives the synthesis of GFP-miR30-shRNA/Luc transcript to induce substantial Luc knock-down.

tTA binds to Tretight promoter in LV-Tretight-GFP-miR30-shRNA/Luc and activates the expression of shRNA/Luc.

In these constructs gene targeting sequences were embedded in the precursor miRNA context derived from miR30, one of the most well-studied miRNA in mammals.

Abbreviation Vector combination Function LVVs-miRLuc-neuroneLV-SYN-Luc+ LV-Tretight-GFP-miR30-shRNA/Luc+ LV-mCMV/SYN-tTA Neurone-specific Luc knock-down system.

LVVs-miRnNOS-neuroneLV-Tretight-GFP-miR30-shRNA/nNOS+ LV-mCMV/SYN-tTA Neurone-specific nNOS knock-down system.

LVVs-miRLuc-control2LV-GfaABC [1]D-Luc+ LV-Tretight-GFP-miR30-shRNA/Luc + LV-GfaABC1D-WPRE Control combination used in Luc knock-down experiments for the astrocyte-specific system.

LVVs-miRnNOS -negative control1LV-Tretight-GFP-miR30-shRNA/Luc+ LV-mCMV/SYN-tTA Negative control combination used in nNOS knock-down experiments for the neurone-specific system.

Again, the anti-Luc construct LV-Tretight-GFP-miR30-shRNA/Luc (treatments 4 in Figure 4c and Figure 4d) did not trigger nNOS knock-down.

LVVs-miRnNOS-control2LV-Tretight-GFP-miR30-shRNA/nNOS + LV-GfaABC [1]D-WPRE Control combination used in nNOS knockdown experiments for the astrocyte-specific system.

LVVs-miRLuc-gliaLV-GfaABC [1]D-Luc+LV-Tretight-GFP-miR30-shRNA/Luc+LV-mCMV/GfaABC1D-tTA Astrocyte-specific Luc knock-down system.

LVVs-miRnNOS-control1 LV-Tretight-GFP-miR30-shRNA/nNOS + LV-SYN-WPRE Control combination used in nNOS knockdown experiments for the neurone-specific system.

LVVs-miRnNOS-glia LV-Tretight-GFP-miR30-shRNA/nNOS + LV-mCMV/GfaABC1D-tTA Astrocyte-specific Luc knock-down system.

Lentiviral systems developed in the course of this study enable tight Dox-controllable and cell-specific miR30 -based RNAi gene knock-down.

LVVs-miRLuc-control1LV-SYN-Luc+ LV-Tretight-GFP-miR30-shRNA/Luc+ LV-SYN-WPRE Control combination used in Luc knock-down experiments for the neurone-specific system.

LVVs-miRnNOS -negative control2LV-Tretight-GFP-miR30-shRNA/Luc+ LV-mCMV/GfaABC1D-tTA Negative control combination used in nNOS knock-down experiments for the astrocyte-specific system.

It is important to note that anti-Luc construct, LV-Tretight-GFP-miR30-shRNA/Luc (treatment 4 in Figure 4a and Figure 4b), was without effect in either cell line, indicating that the nNOS knock-down was sequence-specific.

Figure 3Analyses of the efficacy of miR30-shRNA/Luc in vivo in adult rat brain.

First, the effect of miR30-shRNA/Luc was assessed in cell lines.

To examine whether the different RNAi efficiency in DVC and HIP is caused by different processing of RNAi, we performed northern blotting analysis to assess the ratio between mature -RNAi and precursor-miR30 -RNAi in these two regions.

Analysis of the effects of miR30-shRNA/Luc in vivo.

We first confirmed the efficacy of the anti-nNOS construct, LV-Tretight-GFP-miR30-shRNA/nNOS in PC12 and 1321N1 cells.

Figure 4Western-blot analyses of the functions of miR30-shRNA/nNOS both in vitro (a, b) and in vivo (c, d).

A: LVVs-miRLuc-control1; B: LV-SYN-Luc + LV-Tretight-GFP-miR30-shRNA/Luc + LV-mCMV/GfaABC [1]D-tTA.

To construct the LV-Tretight-GFP-miR30-shRNA/nNOS shuttle vector, we replaced the Luc shRNA sequence in the LV-Tretight-GFP-miR30-shRNA/Luc shuttle vector with the nNOS shRNA.

Figure 2Analyses of the functions of miR30-shRNA/Luc in vitro.

A': LVVs-miRLuc-control2; B': LV-GfaABC [1]D-Luc + LV-Tretight-GFP-miR30-shRNA/Luc + LV-mCMV/SYN-tTA.

To generate the LV-Tretight-GFP-miR30-shRNA/Luc shuttle vector, we excised the Tretight fragment containing the modified Tet-responsive promoter from pTRE-Tight-DsRed2 (Clontech) and inserted it into the pTYF-SW Linker and cloned, into the obtained vector, PCR product of GFP-miR30-shRNA/Luc cassette from pPRIME-CMV-GFP-FF3 (kindly provided by F. Stegmeier, Harvard Medical School) downstream of Tretight promoter.

Our constructs, following the design of Stegmeir et al. used flanking and loop sequences from an endogenous miR30 [37].

Analyses of the functions of miR30-shRNA/Luc in vitro.

3

Here we confirmed that Mtdh represents a target of mi-R30s in DN, and that the expression of all five miR-30 family members is downregulated in the glomeruli form streptozotocin -induced diabetic rats and HG -induced MPC5 cells.

Mtdh protein expression was shown to be increased as well after the treatment with the miR-30 inhibitors (Figure 6e), whereas miR-30 mimics significantly reduced Mtdh expression in HG -induced MPC5 cells (Figure 6f).

To assess the effects of miR-30s on the expression of Mtdh, we transiently transfected MPC5 cells with miR-30 inhibitors, synthetic miRNA mimics, or their NCs.

Furthermore, miR-30 inhibitors significantly increased the expression of Bax and cleaved caspase 3 (Figure 7c).

The obtained results demonstrated that miR-30a, -30b, -30c, -30d, and -30e mimics can significantly inhibit the luciferase activity of the wild-type Mtdh 3′-UTR reporter, but not that of the NC, and that this inhibition was reduced when the mutant reporter, with mutated miR-30 -binding site, was used (Figures 5d–h).

Transient transfections with siRNAs, Mdth overexpression vector, miR-30 inhibitors, and miR-30 mimics.

These cells were transfected with Mtdh siRNA (50 nM; GenePharma, Shanghai, China) or the overexpression (500 ng per well, GenePharma) the mixture of the mimics or inhibitors (RiboBio, Guangzhou, China) of all five miR-30 family members at the final concentrations of 50 nM, using Lipofectamine 2000 (Invitrogen) for 6 h in OPTI-MEM (Gibco BRL), according to the manufacturers' instructions.

Mtdh represents a direct target of the members of miR-30 family.

Mtdh mRNA level was shown to be significantly increased following the treatment with miR-30 inhibitors (Figure 6c), whereas miR-30s mimics led to a considerable reduction of Mtdh expression induced by HG (Figure 6d), compared with the corresponding NC treatment groups.

Therefore, we studied miR-30 expression in the DN glomeruli and HG -induced MPC5 cells.

Afterward, OPTI-MEM was replaced with the complete medium containing 1% FBS, and treated with HG for 48 h after the transfection with siRNAs or mimics, whereas the cells treated with miR-30 inhibitors were not treated with HG.

MPC5 were transfected with miR-30 inhibitors, mimics, or the respective NCs.

analysis demonstrated that the transfection of cells with miR-30 inhibitors significantly increased the percentage of apoptotic cells compared with the NC group (Figure 7a).

[44] The 3′-UTR of Mtdh containing putative miR-30 -binding sites was amplified and cloned into PmiR-RB-REPORT dual-luciferase reporter vector (RiboBio).

The treatment with miR-30 mimics considerably decreased HG -induced increase in the levels of these proteins (Figure 7d).

Conversely, miR-30 mimics considerably reduced the rate of MPC5 apoptosis induced by HG (Figure 7b).

4

Our experiments revealed that expression of the miR-30 family was down-regulated in cardiomyocytes of rats from the TAAC group, and that miR-30a expression decreased in hypertrophic cardiomyocytes that had been treated with Ang II.

Up-regulation of Autophagy and Down-regulation of miR-30 in Rats with TAAC-Induced Cardiac Hypertrophy.

The results of our study indicate that Ang II excessively up-regulates cardiomyocyte autophagy by decreasing miR-30 expression, and that this excessive autophagy promotes the development of myocardial hypertrophy.

Taken together, these results provide strong evidence that autophagy mediates the development of myocardial hypertrophy in cardiomyocytes: a down-regulation of miR-30 induced by Ang II leads to excessive autophagy in cardiomyocytes, thereby promoting myocardial hypertrophy.

A study by Duisters et al. [10] found that miR-30 family members were significantly down-regulated in mouse hypertrophic hearts and in cardiac biopsies from patients with LVH.

The miR-30 family is associated with the development of tumors and other diseases (of the nervous, genital, circulatory, alimentary, and respiratory systems), as well as adipogenesis, cellular senescence, drug metabolism and cell differentiation [16]– [31].

Bioinformatics software predicts that miR-30a may regulate the expression of beclin-1. The function of the miR-30 family is similar to that of other microRNAs.

Down-regulation of miR-30 leads to myocardial hypertrophy.

Consistent with this, the expression level of miR-30 in the plasma of peripheral blood was elevated in patients with left ventricular hypertrophy (LVH).

Autophagy and miR-30 expression in rats after TAAC surgery.

circulating miR-30 expression in rats from the TAAC group.

It was thus of interest to investigate whether the down-regulation of miR-30 might mediate Ang II -induced myocardial hypertrophy.

miR-30-regulated Autophagy in Cardiomyocytes.

Relationship between the circulating miR-30 level and ventricular wall thickness.

Downre-gulation of miR-30 Leads to Myocardial Hypertrophy.

Use of plasma miR-30 levels for the diagnosis of LVH reached statistical significance (P = 0.039, Figure 12B).

Circulating Levels of miR-30 Increased in Rats Following TAAC Surgery, and in Patients with LVH.

5

Furthermore, we also demonstrated that miR-30a-e expression was markedly inhibited in HK-2 (Figure 2g) and NRK-52E (Figure 2h) cells exposed to cisplatin, further confirming the observation in renal tissue in Figure 1. As shown on Figures 1 and 2, miR-30b and miR-30c were expressed stronger than miR-30a, miR-30d and miR-30e in renal tubular cells and tissues.

Interestingly, we discovered that the expression levels of miR-30a, miR-30b, miR-30c, miR-30d and miR-30e were markedly inhibited by cisplatin exposure, especially that of miR-30b, miR-30c and miR-30e as the inhibition was significantly different from that on day 1 of cisplatin injection (Figure 1j).

By comparing the upregulated genes in the cisplatin -induced kidney injury mo dels, we chose putative genes, including Adrb1, Bnip3L, Hspa5 and MAP3K12, as possible target genes of miR-30 s as they were in the intersection of both database queries (Figure 4a).

To elucidate the target genes of miR-30 in cisplatin -induced cell apoptosis, we used bioinformatics tools to predict potential potent target genes.

23, 24 Moreover, we detected the expression of the miR-30 family in renal tissue and found that miR-30c was the highest expressed miRNA among the five members (Figure 1i).

[38] Guo et al. [38] demonstrated that miR-30e, as a member of the miR-30 family, protected against aldosterone -induced podocyte apoptosis and mitochondrial dysfunction by directly targeting Bnip3L.

Although the in vivo evidence about the role of miR-30c on cisplatin -induced renal tubular cell apoptosis lacks, our current experimental results in this study are generally consistent with the observation in which podocyte apoptosis induced by either TGF-beta or puromycin aminonucleoside treatment was ameliorated by exogenously expressing miR-30 and aggravated by the knockdown of miR-30.

[34] Here we revealed that all of the miR-30 miRNAs, including miR-30a, miR-30b, miR-30c, miR-30d and miR-30e, were downregulated in both renal tubular epithelial cells and HK-2 cells when cell apoptosis in renal tubules was induced by cisplatin exposure (Figures 1 and 2).

[36] Roca-Alonso et al. [37] reported that miR-30 overexpression protects cardiac cells from doxorubicin -induced apoptosis.

Using bioinformatics tools, we predicted putative genes, including Adrb1, Bnip3L, Hspa5 and MAP3K12, to be the target genes of miR-30.

All of these reports and our results reveal that the miR-30 family indeed has a regulatory role in the modulation of the cell cycle and cell apoptosis in a variety of pathophysiological processes.

The reason for focusing on this family is that the miR-30 family is the highest abundant miRNA family in renal tubular epithelial cells according to the results of our previous gene microarray.

In this study, we focused on the role of the miR-30 family in the protection of renal tubular cells from the injury induced by cisplatin.

6

miR-30 family was downregulated during pathological hypertrophy to activate calcium signaling, apoptosis and autophagy pathways miR-133b CyclinD, Nelf-A, RhoA, Ccd42 The expression of miR-133 was upregulated during physiological cardiac hypertrophy.

To be specifically mentioned (fold change >2.5), miR-208, miR-19b, miR-133b and miR-30e were significantly upregulated and miR-99b, miR-100, miR-191a, miR-22 andmiR-181a-1 were significantly downregulated.

It increases cell survival and negatively regulates apoptosis by targeting Bcl2 miR-30 CaMKIIδ, Egfr1, Bcl2 miR-30 was significantly upregulated during physiological hypertrophy.

miR-208a, miR-133b and miR-30e showed more than 2.5-fold upregulation during physiological cardiac hypertrophy, while miR-19b showcased an upregulation of 1.5 fold only.

Hence, it is logical to propose that the downregulated expression of miR-30 family in pathological hypertrophy activates calcium signaling, apoptosis and autophagy pathways.

angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy [28].

Our data extend these concepts by demonstrating an upregulation of miR-30 family in physiologically hypertrophied rat heart (Fig. 2A, B).

Previous studies indicate that angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy [28].

7

The antagomirs designed to inhibit the expression of endogenous miR-30 members and Antagomir Negative Control were obtained from Ribobio.

HWJMSC-EVs Deliver and Restore the Expression of miR-30 in Injured Rat Kidney.

Mitochondrial Apoptotic Pathways Are Inhibited by EVs-Derived miR-30.

Meanwhile, antagomir -treated EVs group revealed lower miR-30 expression as well as the vehicle group (Figure 2(c)).

We demonstrated that hWJMSC-EVs may ameliorate acute renal IRI by inhibition of mitochondrial fission via miR-30.

We next explored the miR-30 expression of rat kidney during IRI in vivo.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) showed that IRI caused lower expression of miR-30b, miR-30c, and miR-30d (miR-30a and miR-30e did not exist in rat kidney) and EVs treatment entirely reversed the reduction (Figure 2(a)).

Given that miR-30 has been reported to regulate mitochondrial fission through the DRP1 pathway on cardiomyocytes [20], we hypothesized that human Wharton Jelly MSCs (hWJMSCs) derived EVs may be involved in the modulation of mitochondrial fission via miR-30, thereby protecting kidney from IRI.

To understand how hWJMSC-EVs exert effects on mitochondrial fission, we test whether EVs-derived miR-30 family plays a crucial role in regulating mitochondrial fission through DRP1.

EVs-Derived miR-30 Members Regulate Mitochondrial Fission through DRP1.

To further explore the mechanism of miR-30 reversion, we used specific miR-30 antagomir to treat MSCs.

As we expected, miR-30 antagomir mitigated this effect, especially in miR-30b/c/d antagomir cotreated group.

The miR-30 family is involved in several cellular processes, including cardiomyocytes exposed to oxidative stress or ischemia injury and apoptosis of type II alveolar epithelial cells [20, 32, 33].

Here we have identified a miR-30-related antiapoptotic pathway involving DRP1 and mitochondria, which may be one of the mechanisms by which hWJMSC-EVs alleviate renal ischemia reperfusion injury.

The absence of miR-30 in EVs canceled the miR-30 restoration effects in normal EVs treatment group in vitro.

Then, we treated hWJMSCs with miR-30 antagomir.

The levels of miR-30 family members analyzed by qRT-PCR were normalized to that of U6.

Taken together, it appears that EVs-derived miR-30 can block the mitochondrial apoptotic pathways.

Our research reveals links among EVs, miR-30, and DRP1 in the apoptotic program of the kidney.

Our results suggest that modulation of miR-30 from EVs may represent a therapeutic approach to treat apoptosis-related renal ischemia reperfusion injury.

The absence of miR-30 in EVs canceled the miR-30 restoration effects in normal EVs treatment group.

8

Analysis at P21 revealed 74 mature miRNAs with differential expression between LPD -induced IUGR rat lungs and control lungs (Fig 1): 10 showed more than twofold differential expression: miR-184, miR-127-3p, miR-378a-5p and miR541-5p were downregulated, and miR-30e-5p, miR-23b-5p, miR-451-5p, miR-1839-5p, miR-449a-5p, and miR-19b-3p were upregulated in LPD -induced IUGR versus control lungs.

Furthermore, five deregulated miRNAs (miR-128-3p and miR-34c-5p, both downregulated at P10; miR-19b-3p, miR-449a-5p and miR-30e-5p, these three being upregulated at P21) have E2F3 in common as a target gene.

At P21, the differential expression of 5 of 10 of the miRNAs was statistically confirmed, with miR378a-5p, miR127-3p, and miR184 downregulated and miR30e-5p and miR449a-5p upregulated.

Among the other miRNAs found deregulated in our study, miR-34c-5p, miR-128-3p miR-184, miR127-3p, miR-30e-5p, and miR-23b-5p were also previously described as “tumour suppressors”[22– 26], although many miRNA studies previously dealt with cancer or oncogenic proliferation states and the current analysis depended on previous reports.

9

Interestingly its target gene BDNF, whose expression is compromised in depressed brain [58] and whose expression and functions are regulated by CORT, [59] is predicted to be regulated by miR-124 and miR-30e.

[67] On the other hand, MECP2, a transcriptional regulator targeted by miR-19, miR-30e and miR-365, binds to methylated DNA and has been shown to contribute to early life stress -dependent epigenetic programming of HPA axis -associated genes.

As listed in Supplementary Table 6, these genes were predicted targets of miR-124, miR-101, miR-29a, miR-30e, miR-181c, miR-365 and miR-218.

[63] A large number of cyclic AMP-specific PDEs are also targets of multiple miRNAs (miR-101a, miR-124, miR-721, miR-137, miR-19b, miR-30e, miR-365).

For example, miR-218, miR-324-5p, miR-365 and miR-146a were localized on chromosome 10; miR-764-5p and miR-351 on chromosome X; miR-101 and miR-30e on chromosome 5; miR-582 and miR-137 on chromosome 2; miR-153 and miR-203 on chromosome 6; miR-124 and 181a on chromosome 3 and miR-135a*/miR-135a-3p and let-7i on chromosome 7. Some of the miRNAs that were localized on the chromosome and in close proximity showed the same direction of changes.

10

Consistently, in our study of the roles of miR-30 in regulation of autophagy, we observed that only expression of miR-30c showing around 62 % decrease during activation of autophagy under I/R injury conditions in Fig.   2a.

The result showed no changes in expression of miR-30 except that decreased in miR-30c (Fig.   2a) suggesting an involvement of miR-30c in neuroprotective effect against spinal cord ischemia-reperfusion injury of H2S.

I/R group To explore the mechanisms how H [2]S could attenuate I/R -induced spinal cord injury, we examined the expression of miR-30 profiles by using.

Recent study showed that miR-30 could impair autophagic process by targeting multiple genes in the autophagy pathway [27].

OGD group Recent study showed that miR-30 could impair autophagic process by targeting multiple genes in the autophagy pathway [22].

Fig. 2Expression of microRNA30 profiles and autophagy-related protein in rats spinal cord.

11

Another recent study suggested that down-regulation of miR-30 family leads to endoplasmic reticulum stress in vascular smooth muscle cells [25] and a similar trend of down-regulation is shown in the case of miR-30d and miR-30e.

Indeed, our high confidence network suggests that RUNX2 is controlled by miR-30d as well as miR-30e and both of these miRs are down-regulated which suggests that they play a major role in regulating osteoblast differentiation.

Previous studies have shown that miR-30 family members negatively regulate osteoblast differentiation [24] and also suggest RUNX2 and SMAD1 are common post-transcriptional targets of miR-30 family.

12

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.

13

MiR-107, miR-30 and miR-137 were upregulated only in astrocytes.

Recently it was reported that members of miR-30 family are involved in myocardial extracellular matrix remo deling targeting connective tissue growth factors that are induced by TGF-β and endothelin [79].

J Cereb Blood Flow Metab 79 Duisters RF Tijsen AJ Schroen B Leenders JJ Lentink V 2009 miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remo deling.

To our knowledge, the role of miR-30 has not been studied in astrocytes.

It is tempting to speculate a role of miR-30 in controlling apoptosis in astrocytes under hypoxic stress conditions and in induction of astrocyte proliferation and reactive gliosis through similar pathways as in cardiomyocytes.

14

MiR-30 family members are strongly upregulated during adipogenesis in human cells, and inhibition of miR-30 inhibits adipogenesis [12].

miR-30 family members have also been demonstrated to act as positive regulators of adipocyte differentiation in a human adipose tissue-derived stem cell mo del [35].

The miR-30 family has been found to be important for adipogenesis [12].

15

The underlying mechanism might be related to the modulation of 18 upregulated and 14 downregulated miRNAs, in particular, miR-20a, miR-145, miR-30, and miR-98.

Thus, involution was obtained by miChip analysis for four selected miRNAs that showed either high (miR-145) or low (miR-30) signal intensities, or high (miR-20a) or low (miRNA-98) differential expression values among the three groups.

16

Such a situation occurred for miR-26b, miR-30, and miR-374 downregulation, and for miR-34, miR-301, and miR-352 upregulation [121].

These miRNAs (miR-15a, miR-30, miR-182, and miR-804) are involved in cell proliferation, apoptosis, inflammation, epithelial-mesenchymal transition, invasion, oncogene inhibition, and intercellular adhesion.

17

According to this observation, Caruso et al. (2010) showed that, during the onset of pulmonary arterial hypertension (PAH) after hypoxia, there is a reduced Dicer expression leading to miR-22, miR-30, and let-7f down-regulation and, at the same time, to miR-322 and miR-451 up-regulation in two different PAH rat mo dels.

18

It is noteworthy that miR-1, miR-133, miR-30, miR-208a, miR-208b, mir-499, miR-23a, miR-9 and miR-199a have previously been shown to be functionally involved in cardiovascular diseases such as heart failure and hypertrophy [40], [41], [42], [43], [44], and have been proposed as therapeutic- or disease-related drug targets [45], [46].

Conserved microRNA signatures were identified in valves (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and in ventricular-specific regions of the myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*) of Wistar rat, Beagle dog and cynomolgus monkey.

An assessment of the degree of conservation for structure-specific distribution of microRNAs in Wistar rat, Beagle dog and cynomolgus monkey (see for relative enrichment analysis), revealed high enrichment of nine microRNAs cardiac valves (miR-let7c, mIR-125b, miR-127, mir-199a-3p, miR204, miR-320, miR-99b, miR-328 and miR-744) (Figure 3A) and seven microRNAs in the myocardium (miR-1, mir-133a, miR-133b, miR-208b, miR-30e, miR-499-5p, miR-30e*) (Figure 3A).

19

Finally, other subsets of miRNAs were either up-regulated (miR-23a-3p, miR132-3p, miR-146a-5p, miR-154-3p, miR-181d-5p, miR-212-3p, miR-212-5p, miR-344b-5p, miR-380-3p, miR-410-3p, miR-433-3p and miR-3584; Fig. 2, Supplementary Fig. S4), or down-regulated (miR-29c-5p, miR-30a-5p, miR-30c-2-3p, miR-30e-3p, miR-138-5p, miR-140-3p, miR-551b-3p and miR-652-3p; Fig. 2, Supplementary Fig. S5) during all phases of the disease.

20

Our previous work also revealed that the heart failure of the rat with selenium deficiency was probably related to five upregulated miRNAs (miR-374, miR-16, miR-199a-5p, miR-195 and miR-30e) and three downregulated miRNAs (miR-3571, miR-675 and miR-450a) [11].

Of these miRNAs, such as rno-miR-16, rno-miR-199a-5p, rno-miR-195, rno-miR-30e, which had been reported to be involved in selenium deficiency induced heart failure [11].

21

The expression of miR-101, miR-30b, miR-30c, miR-30d and miR-27b was detected (Figure 1D) but miR-1 and miR-30e expression was not (data not shown).

Six miRNAs (miR-1, miR-101, miR-30b, miR-30c, miR-30d, and miR-30e) were selected to potentially target Sox9 (Figure 1A).

22

It was found to be targeted by multiple microRNAs in our analysis, including miR-143, miR-30 family members, miR-140, miR-27b, miR-125a-5p, miR-128ab, and miR-342-3p.

Among the important genes were Lifr, Acvr1c, and Pparγ which were found to be targeted by microRNAs in our dataset like miR-143, miR-30, miR-140, miR-27b, miR-125a, miR-128ab, miR-342, miR-26ab, miR-181, miR-150, miR-23ab and miR-425.

According to our in silico analysis, Ppar γ is likely regulated by microRNAs like let-7 family members, miR-30 family members, miR-27b, miR-23ab, miR-93, miR-25, miR-128ab, miR-320, and miR-135.

23

In this study, we constructed a PDGFR-β shRNA expression plasmid, in which PDGFR-β shRNA embedded in an miR30 -based context was driven by a CMV promoter.

The PDGFR-β shRNA was inserted immediately into the miR30 -based shRNA expression system of the pCMR30 vector (Genechem, Shanghai, China) to generate a pCMV–shRNA–red fluorescent protein (RFP), in which PDGFR-β shRNA was driven by the CMV promoter and RFP was the reporter gene.

To generate pCMV–shRNA–LacZ, the fragment containing miR30 -based PDGFR-β shRNA was PCR-cloned into the HindIII site of the pMIR-REPORT β-gal reporter control vector (Ambion, Austin, TX, USA), which supplies the β-gal reporter gene.

As a negative control, we generated pCMV–NC–LacZ with miR30 -based PDGFR-β shRNA in pCMV–shRNA–LacZ replaced by negative control RNA.

24

Eleven of the altered miRNAs were downregulated (miR-122, miR-93*, miR-872, miR-7*, miR-146a, miR-342-3p, miR-150, miR-139, miR-30a, miR-30e, miR-320), whereas three miRNAs, namely miR-463*, miR-34c* and miR-1188, were upregulated in the RYGB group.

In addition to miR-342-3p and miR-34c*, miR-872*, miR-463*, miR-30e, miR-2183, miR-1971, miR-150, miR-146a, miR-1188 and miR-93* all correlate with liver energy metabolism processes, such as glycolysis and glycogenesis involving glucose, glycogen and lactate.

25

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).

26

Members of the miRNA-30 family are highly expressed in the kidneys of humans and rats [44] and changes in their expression may result in glomerular disease [45].

27

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.

28

Shen Y. Shen Z. Miao L. Xin X. Lin S. Zhu Y. Guo W. Zhu Y. Z. miRNA-30 family inhibition protects against cardiac ischemic injury by regulating cystathionine-γ-lyase expression Antioxid.

29

The altered regulation of miR-30a is also of interest because the miR-30 family is well studied but has not previously been reported as being involved in vascular changes after SAH.

Duisters RF, Tijsen AJ, Schroen B, Leenders JJ, Lentink V, van der Made I, et al. miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remo deling.

The miR-30 family members are known for their role in angiogenesis-related myocardial matrix remo deling via their interaction with connective tissue growth factor [37].

30

qPCR of left atrial chambers demonstrated that miR-1, miR-26b, miR-29a, miR-30e, miR-106b, miR-133 and miR-200 are up-regulated in HTD rats as compared to controls (Fig 1), demonstrating a similar microRNA expression profile as in atrial-specific Pitx2 deficient mice [14, 16].

31

Disagreements extend to other cell death-related microRNAs that were reported by Liu [6] to demonstrate changes in expression that we were not able to detect (mir-137 and miR-672) or whose expression did not seem to change in the present study (miR-214, miR-30-3p, miR-235-3p, and miR-674-5p).

32

In addition to the aforementioned study of the expression of mir-30d, there were several other expression profiling reports suggesting the involvement of mir-30 family in diabetes or adipogenesis [47]– [49].

33

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.

34

There were no significant changes in the expression of miR-15, miR-30, and miR-133a between healthy subjects and patients with burn injury.

MiR-195, let-7e, miR-15, miR-133a, and miR-30 did not show any significant difference between burn rats and sham rats (Figure 1A) (P>0.05).

MiR-15, miR-133a, and miR-30 also had P [CT] > 0.75.

35

Previous studies have demonstrated that angiotensin II could induce the down-regulation of miR-30 [41], increase mitochondria oxidative stress [42], thereby causing myocardium autophagy, which indicated that RAS activation could result in the cellular autophagy.

36

miR-30 regulates mitochondrial fission through targeting p53 and the dynamin-related protein-1 pathway.

37

As previously reported [14], short hairpin RNAs targeting firefly luciferase (accession number M15077, non-mammalian control, luciferase shRNA), rat VEGF [164] (accession number AF260425, VEGF [164] shRNA) and rat VEGFA (accession number NM_031836, VEGF-A shRNA) were embedded into a microRNA (miR-30) context.

The miR-30/shRNAs were cloned into a lentiviral transfer vector under transcriptional control of a CD44 promoter (pFmCD44.1 GW), with green fluorescent protein (GFP) as a reporter gene.

38

Pan et al. reported that miR-30-regulated autophagy mediates angiotensin II -induced myocardial hypertrophy [44], which implies that there exists a complicated network for mediating autophagy that has yet to be determined.

It should be noted that the miR-352-IGF2R pathway may not be the only mechanism for regulating autophagy during collateral vessel growth because additional miRNAs involved in autophagy were identified, such as, miR-30.

39

Nonetheless, the authors identified a pool of dysregulated miRNAs (∼50 miRNAs) in which some of them such as miR-144, miR-106b, miR-185, miR-30e, miR-589, miR-665 and many more showed similarities in expression as observed in our study (either humans/animals or both).

40

PE + miR30 inhibitor.

PE + miR30 mimics, [&&]p < 0.01 vs.

41

Column 4 is a similar co transfection with another shRNA vector based on the mir-30 system that contains the same shRNA targeting sequence as the shRNA shown in column 3 experiments.

42

No differences in miRNA expression were found in kidneys of ischemic heart failure mice compared to control animals (S6 Table) and only small differences were observed between expression levels of miR-18a-5p, miR-30e-5p, miR-199a-3p and miR-223-3p in the LV of mice with ischemic heart failure compared to controls (S7 Table), however not reaching significance after Bonferroni correction for multiple testing.

43

The template “miRNA30‐like” DNA oligonucleotides targeting two positions of NR1 mRNAs (shRNAmir1 and shRNAmir2) were ligated into the XhoI/ MluI sites in the pGIPZ‐shRNAmir vector (Open Biosystems).

44

The reference genes utilised were miRNAs with stable expression across samples according to BestKeeper: miR-22, miR-30a, miR-30c, miR-30e and miR-100.

45

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.

46

Out of the 14 miRNAs that were detected to be differentially expressed using NGS (Table  4), five (rno-let-7d-5p, rno-miR-100-5p, rno-miR-203a-3p, rno-miR-21-5p, rno-miR-320-3p) were represented on the TLDA-A, while nine (rno-miR-378a-3p, mmu-miR-5100, rno-miR-30e-3p, rno-miR-125b-2-3p, rno-miR-320-5p, rno-miR-3473, rno-miR-21-3p, rno-miR-455-5p, and hsa-miR-7641) were not.

47

The target miRNAs (and corresponding assay numbers) were: rno-miR-23a (000399), rno-miR-26b (000407), rno-miR-30-5p (000420), rno-miR-101b (002531), rno-miR-125b-5p (000449), rno-miR-379 (001138) and rno-miR-431 (001979).

48

We designed and cloned into the pcPURhU6 vector the hairpin-type RNAs with si-6 sequence (pcPURhU6 si-6) with the 19-21 base pair (bp) stems and with various loops: (1) pcPURhU6 si-6 (21 bp)-miR26, (2) si-6 (19 bp) with 9-nt UUCAAGAGA loop [28], (3) si-6 (21 bp) with 9-nt UUCAAGAGA loop, (4) si-6 (21 bp) with 10-nt CUUCCUGUCA (loop from miRNA23), and (5) si-6 (21 bp) with 19-nt UAGUGAAGCCACAGAUGUA (loop from miRNA30) (see Figure 3).

Constructs 4 and 5 with miRNA-origin loops miRNA23 and miRNA30, respectively, demonstrated moderate silencing activity, lowering BACE1 mRNA by 27% and 38%, respectively.

49

Micro RNAs quantified were hsa-miR-25-3p (Cat# 204361), mmu-miR-155-5p (Cat# 205930), hsa-miR-99b-3p (Cat# 204064) and mmu-miR-451(Cat# 204734) and normalized with RNU5G (a small nuclear RNA) in heart and hsa-miR-30e-5p (Cat# 204714) in plasma.

D), E) Relative levels of miR-25, miR-155, miR-451 and miR-99b in heart normalized with sn-RNU5G; F) Relative levels of miR-25, miR-451 and miR-99b in plasma normalized with miR-30e.

50

Moreover, the fine coordination of activity of the transmembrane receptor smoothened by the miR-30 family allows the correct specification and differentiation of distinct muscle cell types during zebrafish embryonic development 41.

51

Duisters R. F. Tijsen A. J. Schroen B. Leenders J. J. Lentink V. van der Made I. Herias V. van Leeuwen R. E. Schellings M. W. Barenbrug P. miR-133 and miR-30 regulate connective tissue growth factor: Implications for a role of micrornas in myocardial matrix remo deling Circ.

52

The amiRNAs represent a type of shRNAs in which a siRNA sequence is embedded into a native microRNA scaffold [30], most commonly into that of miR-30 [30], [31] or miR-155 [32], [33].

We confirmed that the silencing efficiency of amiR155-PLBr used here was comparable to that using a miR-30 scaffold (data not shown).

53

Duisters R. F. Tijsen A. J. Schroen B. Leenders J. J. Lentink V. van der Made I. Herias V. van Leeuwen R. E. Schellings M. W. Barenbrug P. miR-133 and miR-30 regulate connective tissue growth factor: Implications for a role of microRNAs in myocardial matrix remo deling Circ.

54

The authors also demonstrated that resilient rats differed from vulnerable rats in the set of multiple blood-circulating miRNAs, namely, reduction in miR-139-5p, miR-28-3p, miR-326-3p, and miR-99b-5p in resilient animals and reduction in miR-24-2-5p, miR-27a-3p, miR-30e-5p, miR-3590-3p, miR-362-3p and miR-532-5p levels in vulnerable animals.

55

All short hairpin RNAs (shRNAs) were generated using a mir-30 -based design method, which has been previously described [25].

56

Interestingly, among the conserved miRNAs found in all epididymal regions, we identified 8/14 and 4/7 members of the let-7 family (let-7a—let-7f, let-7i) and miR-30 (miR-30a— miR-30d) family, respectively.

57

MiR-30 was shown to induce apoptosis [32] and regulate cell motility by influencing the extracellular matrix remo deling process [33– 35].

58

Lentiviral vectors encoding miR-20a or a control sequence within a common miR-30 backbone have been previously described [16].

59

These include rno-miR-195, rno-miR-125a-5p, rno-let-7a, rno-miR-16, rno-miR-30b-5p, rno-let-7c, rno-let-7b, rno-miR-125b-5p, rno-miR-221, rno-miR-222, rno-miR-26a, rno-miR-322, rno-miR-23a, rno-miR-191, rno-miR-30 family, rno-miR-21, rno-miR-126, rno-miR-23b, rno-miR-145 and rno-miR-494.