53 publications mentioning rno-mir-210

Open access articles that are associated with the species Rattus norvegicus and mention the gene name mir-210. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

Bioinformatics analysis for miR-210 targets in databases (miRWalk 2.0) and our recent study [28] showed that GR was a potential downstream target of miR-210, implicating that, in addition to promoter hypermethylation, hypoxic -induced upregulation of miR-210 may also contribute to the downregulation of GR expression in cardiomyocytes in response to hypoxia.

Thus, miR-210 suppressed the Firefly luciferase gene expression by binding to the miR-210 target region of GR mRNA 3′-UTR, indicating that the GR transcript is a valid target of miR-210.

Given that HIF-1α played a critical role in the regulation of hypoxia -induced miR-210 expression (Figure 2D), we further determined the role of HIF-1α in hypoxia -mediated suppression of GR protein expression.

normoxia in (B); n = 6. Given that HIF-1α played a critical role in the regulation of hypoxia -induced miR-210 expression (Figure 2D), we further determined the role of HIF-1α in hypoxia -mediated suppression of GR protein expression.

And miR-210 inhibition by its locked nucleic acid (LNA) inhibitor reversed hypoxia -induced suppression of GR protein in primary fetal cardiomyocytes.

Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts.

Furthermore, we identify the GR as a downstream target of miR-210, demonstrating a negative regulation of miR-210 on the expression of the GR by binding to the 3′-UTR of its transcript.

Second, we also provide the novel evidence that GR mRNA is a direct downstream target of miR-210 and that hypoxia suppresses the GR in a HIF-1α- and miR-210 -dependent manner in the fetal rat heart.

We demonstrate that hypoxia reduces GR expression through a miR-210 -dependent pathway, revealing a pivotal role for miR -mediated regulation in epigenetic programming of gene expression patterns in the heart.

normoxia in (B); n = 6. To determine the influence of miR-210 on GR expression under hypoxic conditions, cardiomyocytes were treated with miR-210-LNA or negative control (50 nM) under normoxic or hypoxic (1% O [2]) conditions for 24 hours followed by detection of GR protein expression by Western blot.

Hypoxia induces miR-210 expression and suppresses GR abundance in fetal rat hearts.

To assess the role of HIF-1α in hypoxia -induced miR-210 expression in cardiomyocytes, we administered 2ME, an inhibitor of HIF-1α nuclear accumulation [29, 30], during ex vivo hypoxic treatment in primary fetal cardiomyocytes.

pmiRGLO-XGRX + 7 nM scramble; n = 6. To further determine the inhibitory effect of miR-210 on endogenous GR expression in cardiomyocytes, primary fetal cardiomyocytes were treated with miR-210 mimic or negative control (100 nM) under normoxic conditions for 48 hours followed by detection of GR protein abundance by Western blot.

For transfection with miR-210 inhibitor, cells were cultured in antibiotic-free M199 supplemented with 10% FBS for 1 hour, and then transfected with 50 nM miR-210 locked nucleic acid (miR-210-LNA; Exiqon) inhibitor or its negative control (Exiqon, Woburn, MA) under normoxic (21% O [2]) or hypoxic (1% O [2]) conditions for 24 hours.

normoxia in (B); n = 6. To determine the influence of miR-210 on GR expression under hypoxic conditions, cardiomyocytes were treated with miR-210-LNA or negative control (50 nM) under normoxic or hypoxic (1% O [2]) conditions for 24 hours followed by detection of GR protein expression by Western blot.

miR-210 suppresses GR expression in cardiomyocytes under normoxic or hypoxic conditions.

This finding suggests that site 4 may be an important repressor of miR-210 expression, which restricts miR-210 overexpression in response to hypoxia.

Thus, luciferase assays were used to determine the effect of miR-210 in downregulating GR expression in the rat cardiomyocyte cell line H9c2.

Moreover, the functional significance of negative regulation of GR expression by miR-210 was demonstrated in primary fetal cardiomyocytes showing that miR-210 mimic treatment significantly reduced GR protein abundance.

Taken together, our data indicate that HIF-1α works through miR-210 activity regulating GR expression under hypoxic conditions.

In contrast, cells that were co -transfected with pmiRGLO-XGRX plus miR-210 mimic (pmiRGLO-XGRX + 7 nM miR-210 mimic) had 30% inhibition of luciferase activity compared to cells co -expressing the GR 3′-UTR and a scrambled mimic (pmiRGLO-XGRX + 7nM scramble) (Figure 3B).

Thus, despite deletions of HRE1 and HRE2 motifs, the miR-210 promoter activity in hypoxia remained upregulated to at least 13 fold.

For experimental validation of the miR-210-3p target in rat GR 3′-UTR, H9c2 cells were transfected with 500 ng/well empty pmiRGLO (pmirGLO control vector), pmiRGLOXGRX construct only, pmiRGLOXGRX plus 7 nM miR-210 mimic (miR-210) (Qiagen) or 7 nM scrambled miRNA mimic (Qiagen) plus Attactene transfection reagent (Qiagen) following methodologies as described by Qiagen.

This demonstrates that HRE3 also drives baseline endogenous expression of miR-210 in cardiomyocytes.

These findings are consistent with previous reports that HIF-1α drives miR-210 production under hypoxic conditions as well as baseline miR-210 expression under normoxic conditions in cancer cells [37, 40, 41] and in differentiating myoblasts [36].

In summary, the present study provides insights into the epigenetic mechanisms through which fetal hypoxia alters GR expression in cardiomyocytes by miR-210.

We found that HIF-1α inhibition blocked miR-210 production in response to hypoxia in cardiomyocytes.

Furthermore, we presented novel evidence that maternal hypoxia during gestation induced miR-210 expression in fetal rat hearts, implicating miR-210 as a possible mediator of fetal stress -mediated programming in the heart.

The result showed that miR-210 mimic significantly reduced reporter activity in the cells containing the target sequences of GR 3′UTR.

These data indicate that HIF-1α is an essential determinant of miR-210 expression under normoxic conditions as well as in response to hypoxia.

As shown in Figure 1B, hypoxia stimulated a 14-fold increase in miR-210 expression in fetal cardiomyocytes.

Transfection of either miR-210 mimic or inhibitor was carried out using the HiPerFect transfection reagent (Qiagen Inc.

Moreover, we found that deletion of site 4, which is not a HIF-1α but a myc -associated zinc finger protein binding site, resulted in the upregulation of hypoxia -induced miR-210 level, as compared to cells with intact miR-210 promoter.

MiR-210 expression was determined by miScript miR real-time RT-PCR.

Our recent study provided the first evidence that miR-210 repressed GR expression in the neonatal brain of rats after hypoxic-ischemic brain injury [28].

To validate that miR-210 negatively regulates GR expression, we conducted in vitro UTR analyses using luciferase reporter gene assays.

These findings, together with the substantial body of evidence regarding the cardioprotective effects of glucocorticoids in cardiac ischemic injury, suggesting a potential role of miR-210 in hypoxia -induced fetal programming of the heart by modulating GR expression.

Inhibition of HIF-1α nuclear accumulation abolished miR-210 production in response to hypoxia (Figure 2D).

In the present study, we reveal the role of HIF-1α -dependent miR-210 -mediated suppression of GR in cardiomyocyte cell death in response to hypoxia.

Consistent with previous studies in neonatal rat cardiomyocytes [31], we found that hypoxia induced miR-210 expression in fetal rat cardiomyocytes.

To confirm the role of HIF-1α in the miR-210 -mediated suppression of the GR during hypoxia, we inhibited HIF-1α nuclear accumulation using 2ME and measured GR protein in cardiomyocytes.

Hypoxia -induced miR-210 expression depends on HIF-1α activity.

We then determine the promoter-level regulation of miR-210 in rat cardiomyocytes in response to hypoxia.

We report that hypoxia drives miR-210 production in a HIF-1α -dependent manner, and that this event is directed by a single hypoxia response element (HRE) within the miR-210 promoter.

miR-210 directly binds to the 3′-UTR of GR mRNA.

Thus, it is highly likely that the mutation of complementary nucleotides at GR 3′UTR will abrogate binding and function of miR-210 with regards to GR mRNA.

Taken together, these findings uncovers a new mechanism of GR regulation by miR-210 in hypoxia -induced programming of ischemic-sensitive phenotype in the developing heart.

Thus, all this evidence indicates that miR-210 is a negative regulator of GR in cardiomyocytes through interaction with the 3′-UTR of GR transcript.

Maternal hypoxia caused a 59% increase (p < 0.05) in miR-210 expression in fetal rat hearts compared to fetal hearts from normoxia control animals (Figure 1A).

These studies suggest that the miR-210 promoter is highly conserved across species among humans and rodents.

Interestingly, 2ME also suppressed miR-210 production under normoxic conditions by 77% compared to vehicle -treated cells, which is consistent with the finding of the reporter gene assays of miR-210 promoter activity.

The activity of miR-210 has been linked to the mitochondrial metabolism, angiogenesis, cell proliferation, cell differentiation, apoptotic cell death, etc [25– 27].

Our previous study has determined that miR-210-LNA reduces brain miR-210 levels in a perinatal HI rat mo del [28].

Cloning of rat miR-210 promoter.

Figure 1(A) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O [2] from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR.

MiR-210 is directly linked to hypoxia through its promoter that is driven by nuclear accumulation of hypoxia-inducible factor (HIF)-1α.

In addition, miRbase describes the sequence information of the rat (rno) pre-miR-210 stem-loop and mature miR-210 (http://www.

Function of HREs in miR-210 promoter activity.

The mature miR-210 has significant sequence complementarity of its seed region at the 5′ end (nucleotides 2–8) to the 3′UTR of GR mRNA with an exact match of positions 3–7 in the mature miR-210 to GR 3′UTR.

Deletions of HRE1 and HRE2 resulted in no significant loss of miR-210 promoter activity in hypoxia.

Our findings are in agreement with the previous findings in mouse and human showing that miR-210 promoter harbors three HREs and the HRE3 is identified as the HIF-1α binding site responsible for the robust induction of miR210 promoter activity [36, 37].

In the rat, the miR-210 coding gene is located on chromosome 1. Using the Genbank and miRbase as our points of reference we were able to locate the promoter region of miR-210 on rat chromosome 1. A bioinformatics (Genomatix) transcription factor search of the 998-bp segment upstream of rat pri-miR-210 transcription start site revealed three potential hypoxia response elements (HREs) at −466, −402, −63 (Supplementary Figure 1; Figure 2A).

The sequence information of the rat miR-210 promoter was obtained from Genbank (http://www.

In the present study, we demonstrated that the three putative HREs of rat miR-210 are of the HBS type and HIF-1α is capable of binding to all three HRE sites identified within the miR-210 promoter.

As a result, there was no significant difference between miR-210 promoter activities under normoxic versus hypoxic conditions when HRE3 was deleted.

The mature miR-210 has significant sequence complementarity of its seed region to a single discrete nucleotide sequence in the 3′-UTR of GR mRNA (Figure 3A).

This finding possibly indicates a de-repression of miR-210 promoter activity that is induced when site 4 is deleted.

vehicle in normoxia; n = 7. The relative contribution of each of the three HRE motifs in the regulation of miR-210 promoter activity was tested by site specific deletions (Δ1 through D4) in reporter gene assays along with the WT-miR-210 pGL3; in normoxic and hypoxic (1% O [2]) environments for 26 hours in parallel.

Figure 2(A) Schematic representation of the miR-210 promoter, indicating putative binding sites of HIF-1α/ARNT dimer in three HRE motifs (1, 2 and 3).

The WT-miR-210-pGL2 promoter-reporter construct and all the four deletion constructs were prepared endotoxin free and sequences were confirmed.

Quantitative RT-PCR for GR or miR-210 levels.

For transfection with miR-210 mimic, cells were cultured in antibiotic-free M199 supplemented with 10% FBS for 1 hour, and then transfected with 100 nM miR-210 mimic or its negative control (Qiagen) under normoxic conditions for 48 hours.

MiR-210 locked nucleic acid (miR-210-LNA) were purchased from Exiqon (Woburn, MA).

MiR-210 directly binds to the 3′-UTR of GR mRNA.

vehicle in normoxia; n = 7. (A) Schematic representation of the miR-210 promoter, indicating putative binding sites of HIF-1α/ARNT dimer in three HRE motifs (1, 2 and 3).

Figure 3(A) Schematic illustrating sequence complementarity of the seed sequence of rat miR-210 to the 3′-UTR of GR mRNA.

Bio-informatics (Genomatix) analyses of this miR-210 promoter region identified three putative HIF-1α/ARNT heterodimer binding sites or HREs with core elements of these HREs at -446 (HRE1), -402 (HRE2), -63 bp (HRE3), and a fourth site with core element at -7 where Myc -associated zinc finger protein related transcription factor binds (Supplementary Figure 1).

pmiRGLO-XGRX + 7 nM scramble; n = 6. (A) Schematic illustrating sequence complementarity of the seed sequence of rat miR-210 to the 3′-UTR of GR mRNA.

These findings suggest that of the three HREs, HRE [-63] is the sole determinant of miR-210 promoter activity in response to hypoxic insult in cardiomyocytes.

Because of its robust response to hypoxia and its role in mediating multiple physiological processes, miR-210 has been termed the “master hypoxamir” [25].

Isolated RNA was converted to cDNA using the SuperScript III First-Strand Synthesis SuperMix (Life Technologies) for GR mRNA detection, or miScript II RT kit (Qiagen) for miR-210 detection according to the manufacturer's instructions.

Among these, the most consistent and powerful response to hypoxia is observed in miR-210 activity.

Hypoxia induced a robust increase in miR-210 promoter activity in cells containing the intact wild type miR-210 promoter.

Taken together, we conclude that HIF-1α binding to HRE3 in the miR-210 promoter is the major determinant of miR-210 production in cardiomyocytes, both under normoxic and hypoxic conditions.

A 223 bp segment of 3′-UTR of rat GR mRNA harboring the prospective target region of mature miR-210-3p was first PCR amplified from normal rat heart cDNA using the fo0rward (5′-gagacccCTCGAGggctagacacccattttcaca) and reverse (5′-gagacccTCTAGAgggctactactgcttctgttttg) primers designed based on the rat GR mRNA sequence (accession #: M14053.1) but containing artificial XhoI (CTCGAG) and XbaI (TCTAGA) sites in forward and reverse primers respectively to facilitate cloning.

2

Expression of miR-210 Was Overexpressed or Suppressed by Respective Mimic or Inhibitor Transfection.

The underlying molecular mechanism for miR-210 in regulating cell proliferation, apoptosis and migration was complicated, as miR-210 overexpression protected PC-12 cells against hypoxia -induced injury via suppression of BNIP3 expression, involving the PI3K/AKT/mTOR signal pathway.

Cell proliferation, migration and invasion were inhibited, but apoptosis was promoted, due to hypoxia, could be alleviated by the overexpression of miR-210, while it could be aggravated by miR-210 suppression.

These results suggested that miR-210 was suppressed or overexpressed by treatment with miR-210 inhibitor or miR-210 mimic, respectively.

suggested that miR-210 overexpression alleviated the injury of PC-12 cells induced by hypoxia via suppressing BNIP3 expression and activating the PI3K/AKT/mTOR signal pathway.

Subsequent experiments showed that both mRNA and protein expression of BNIP3 were significantly suppressed (P < 0.05 or P < 0.001) in the miR-210 mimic group of cells, and were significantly increased (P < 0.01 or P < 0.001) in the miR-210 inhibitor group of cells (Figures 5A–C).

The suppression of BNIP3, directly regulated by miR-210 overexpression, may partially contribute to the protection against hypoxic injury in PC-12 cells.

in our study found the effects of miR-210 overexpression on cell viability, migration, invasion and apoptosis were all reversed by BNIP3 overexpression in hypoxia -treated PC-12 cells, suggesting miR-210 functioned through negative regulation of BNIP3.

Figure 3 demonstrated that the expression level of miR-210 in the miR-210 mimic group was significantly higher than the scramble group of cells (P < 0.001), while there was significant decrease in the expression level of miR-210 in the miR-210 inhibitor group compared to the NC group of cells (P < 0.01).

In addition, Bcl-2 was remarkably down-regulated (P < 0.001) while Bax, cleaved/pro caspase-9 and cleaved/pro caspase-3 were significantly up-regulated (P < 0.01) in the hypoxia+miR-210 mimic+pc-BNIP3 group of cells compared to the hypoxia+miR-210 mimic+pcDNA3.1 group of cells (Figures 6H,I).

As a target of HIF-1α, miR-210 is the only miR that is consistently up-regulated in normal and transformed cells, which are exposed to hypoxia, in all the literatures reported previously (Biswas et al., 2010; Chen et al., 2010).

Western blot results of apoptosis related factors showed Bcl-2 was significantly up-regulated while Bax, cleaved/pro caspase-9 and cleaved/pro caspase-3 were all down-regulated in the hypoxia+miR-210 mimic group compared to the hypoxia+scramble group (Figure 4E, P < 0.01 or P < 0.001).

suggested that miR-210 overexpression alleviated hypoxia -induced PC-12 cell injury by down -regulating BNIP3 expression.

The results revealed significant up-regulation of miR-210 expression levels in hypoxia -treated PC-12 cells compared to control group of cells (P < 0.01, Figure 2).

BNIP3, Bcl-2 adenovirus E1B 19 kDa-interacting protein 3; NC, negative control of miR-210 inhibitor; BNIP3-WT, pMiR-report vector carrying wild-type BNIP3 3′-untranslated region (3′UTR) containing miR-210 -binding sites; BNIP3-Mut, mutant BNIP3-WT.

A marked increase in PC-12 cell viability was caused by miR-210 mimics compared to the scramble group of cells (Figure 4A, P < 0.05), while cell viability was significantly decreased following suppression of miR-210 expression as in the hypoxia+miR-210 inhibitor group compared to the hypoxia+NC group (P < 0.05).

Considering that both miR-210 and BNIP3 are pivotal targets of HIF-1, the discovery of miR-210-BNIP3 contributes to the regulatory network of HIF-1 under hypoxia in PC-12 cell, and the network in other cell types needs more experiments.

miR-210 Functioned by Down-Regulating BNIP3.

Previous studies demonstrated that miR-210 was up-regulated in NPCs cultured under hypoxic conditions (Wang et al., 2013).

Moreover, the HI -induced neuronal death was decreased and long-term neurobehavioral function recovery was improved by the down-regulation of miR-210, supporting a detrimental role of miR-210 in neonatal HI brain injury (Ma et al., 2016).

Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression.

Hypoxia-inducible mir-210 regulates normoxic gene expression involved in tumor initiation.

The involvements of miR-210 overexpression, induced by hypoxia, in the regulation of BNIP3 expression in PC-12 cells were investigated.

Hypoxia Up-Regulated miR-210.

In addition, co-transfection of BNIP3-WT and miR-210 mimic showed significant down-regulation of luciferase activity compared with co-transfection of BNIP3-WT and scramble miRs (Figure 5D, P < 0.05), whereas comparison between co-transfections with BNIP3-Mut was non-significant, supporting that the miR-210 mimic could directly bind to BNIP3 3′UTR.

In addition, we interestingly figured out that miR-210 mimics could ameliorate hypoxia -induced cell injury of PC-12 cells, whereas silencing of miR-210 expression leads to the opposite outcome.

miR-210 mimic, scramble miRs, miR-210 inhibitor and its negative control, which was referred to as NC, were synthesized by GenePharma Co.

PC-12 cells were transfected with miR-210 mimic, miR-210 inhibitor or their negative controls.

The study demonstrated that HI led to significant increase of miR-210 expression in neonatal rat brains.

Figure 5BNIP3 is a target of miR-210.

This study is the first to report BNIP3 as a target of miR-210 in PC-12 cells under severe hypoxia, which was consistent with that reported in NPCs previously.

The wild-type BNIP3 3′UTR sequence containing a putative miR-210 -binding site, predicted using online TargetScan software [1] and miRBase database [2] (Chung et al., 2015), was sub-cloned into pMiR-report vector (Ambion, Austin, TX, USA), and the resultant plasmid was referred to as BNIP3-WT.

Neuroprotective effects of microRNA-210 against oxygen-glucose deprivation through inhibition of apoptosis in PC12 cells.

miR-210 suppresses BNIP3 to protect against the apoptosis of neural progenitor cells.

Similar to these results, our study demonstrated the abnormal expression of miR-210 in PC-12 cells under hypoxia.

Inhibition of microRNA-210 provides neuroprotection in hypoxic-ischemic brain injury in neonatal rats.

Hence, BNIP3 was a target of miR-210.

Screen utilizing online TargetScan software and miRBase database revealed the putative binding between miR-210 and BNIP3 3′UTR.

To understand the miR mechanisms in controlling cellular behavior, we used a Bioinformatics method to virtually screen the possible targets of miR-210.

The data presented in this study revealed that miR-210 was overexpressed in hypoxia -induced PC-12 cells.

Mutation at the miR-210 -binding site of BNIP3-WT, which was referred to as BNIP3-Mut, was formed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).

Our study demonstrated that aberrantly expressed BNIP3, which might be modulated by miR-210, could affect the activation of the PI3K/AKT/mTOR signaling pathway in PC-12 cells under hypoxia.

NC, negative control of miR-210 inhibitor.

miR-210 Targeted BNIP3.

In summary, we report that miR-210 protect PC-12 cells against hypoxia -induced injury by targeting BNIP3.

Bcl-2, mammalian B cell lymphoma-2; Bax, Bcl-2 -associated X protein; C/P-, cleaved/pro; NC, negative control of miR-210 inhibitor.

miR-210 Overexpression Alleviated the Injury Induced by Hypoxia in PC-12 Cells.

Among them, miR-210 is a master miR of the hypoxia response, belonging to a specific group of miRs termed “Hypoxamirs” which are regulated by hypoxia (Ma and Zhang, 2017).

We then determined whether miR-210 regulated the injury induced by hypoxia in PC-12 cells.

In our study, we focused on the interaction between BNIP3 and miR-210, presenting that BNIP3 could be negatively regulated by miR-210.

Cells were co -transfected with BNIP3-WT or BNIP3-Mut and miR-210 mimic or scramble miRs using Lipofectamine 3000 reagent.

The results suggested a possible cytoprotective effect of miR-210 on survival of brain cells.

miR-210 has been implicated in neural cell death and pathogenesis of neonatal HI brain injury in a recent study by Ma et al. (2016).

A recent study has reported cell survival and autophagy were promoted by miR-210 (Xu et al., 2016).

Although the interaction between miR-210 and BNIP3 has been reported in the hypoxia -treated NPC cells previously, we verified the links between miR-210 and BNIP3 in another neural cell line, PC-12 cells.

Hypoxia responsive miR-210 promotes cell survival and autophagy of endometriotic cells in hypoxia.

The percentage of apoptotic cells was decreased significantly in the hypoxia+miR-210 mimic group as compared to the scramble group (P < 0.05) while it was increased markedly in the hypoxia+miR-210 inhibitor group compared to hypoxia+NC group (Figure 4D, P < 0.01).

To investigate the expression of miR-210 in hypoxia -treated PC-12 cells, quantitative real-time PCR (qRT-PCR) was carried out.

Relative expression was calculated on the basis of the 2−ΔΔC [t] method (Livak and Schmittgen, 2001), normalizing to U6 (miR-210) or GAPDH (BNIP3 mRNA).

Huang et al. (2009) once identified the promoter region of miR-210 contains a functional hypoxia-responsive element (HRE).

Mounting evidence supports diverse functions of miR-210 in multiple biological processes associated with hypoxia, for example, cell cycle, proliferation, angiogenesis, apoptosis, DNA damage repair, mitochondrial metabolism, differentiation and tumor growth.

Cell apoptosis of HeLa cells has been proved to be elevated by the miR-210 blockade (Cheng et al., 2005).

Bioinformatics methods also identified the putative complementary connection between miR-210 and BNIP3.

Moreover, the downstream signaling pathways besides the interaction between miR-210 and BNIP3 in hypoxia -treated cells are reported for the first time.

Thus, elucidating the precise details between miR-210 and BNIP3 are of great importance for the study of neuroprotection.

The previous literature has reported the potential interactions between miR-210 and BNIP3 in neural progenitor cells (NPCs; Wang et al., 2013).

3

Real-time PCR revealed that Col2a1 expression was significantly upregulated in the inner meniscus cells, and immunocytochemistry also demonstrated that miR-210 could enhance the expression of collagen type 2 in the meniscus cells (P < 0.05, n = 6 for each group) (Figure  5A,B).

In their research, collagen type 1 expression was upregulated in the injured ligament after the administration of miR-210, although it was unclear whether collagen type 1 production occurred as a direct or indirect effect of miR-210.

miR-210 upregulated Col2a1 expression in the inner meniscus cells and VEGF and FGF2 expression in the synovial cells.

In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells.

VEGF and FGF2 expression in synovial cells were upregulated by overexpression of miR-210.

Figure 6 Gene expression analyses in synovial cells after overexpression of miR-210.

This indicated the direct effect of miR-210 on upregulation of collagen type 2 in inner meniscus cells.

In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control.

For gene expression analysis in the meniscus, mature miR-210, Col1a1, collagen type 2 alpha 1 (Col2a1), VEGF and FGF2 expression was examined using real-time PCR.

Type 2 collagen expression was observed around the injured site of the meniscus in the miR-210 group, while its expression was sparse in the control (Figure  4B).

The expression levels for each gene were assessed relative to the expression of snoRNA-135 for miR-210, and relative to ACTB for other genes.

These results demonstrated that miR-210 could enhance the expression of VEGF and FGF2 expression in synovial cells.

Figure 5 Gene expression analyses in inner meniscus cells after overexpression of miR-210.

An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.

In their study, VEGF and fibroblast growth factor (FGF)-2 were upregulated in the injured ligament by administration of miR-210, which also contributes to acceleration of ligament healing.

These results demonstrate that miR-210 could enhance the expression of collagen type 2 in the meniscus cells.

miR-210 plays a role in the induction of VEGF expression, which is recognized as harmful for articular cartilage.

Overexpression of miR-210 has also been reported to stimulate the formation of capillary-like structures in vitro in normoxic conditions as well as vascular endothelial growth factor (VEGF) -driven cell migration [25- 27].

It has been reported that miR-210 is a crucial molecule of the endothelial cell response to hypoxia and that the targets of miR-210 are ephrin-A3, E2F3, MN, ACVR1B, NPTX1, RAD52, CASP8AP2, FGFRL1 and HOXA-1/9, which have important functions in cell survival, migration and differentiation [25, 36- 43].

Hu and colleagues demonstrated that local injection of ds miR-210 generated in a minicircle vector into the myocardium can improve angiogenesis, inhibit apoptosis and improve cardiac function in mice [44].

In our results, high expression of VEGF and FGF2 was observed in vivo after intra-articular injection of miR-210.

There was no significant difference in miR-210, Col2a1, VEGF and FGF2 expression between the control and normal groups (* P < 0.05, N. S. (no significant difference), n = 5 for each group).

The present study demonstrates that intra-articular injection of ds miR-210 can promote the healing of damaged white zone meniscus through the promotion of collagen type 2 production in meniscus cells and through the enhancement of VEGF and FGF2 expression in synovial cells with angiogenesis and cell proliferation.

Expression of miR-210, Col2a1, VEGF and FGF2 in the miR-210 group was significantly higher than that in the control and normal groups (* P < 0.05).

Expression of mature miR-210, collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) was examined using real-time PCR.

To examine which cell type in the meniscal lesion contributed to the meniscal healing by the miR-210 injection, overexpression of miR-210 was conducted in isolated inner meniscus cells or synovial cells in rats in vitro.

The expression of miR-210, Col2a1, VEGF and FGF2 in the miR-210 group was significantly higher than that in the control and normal groups (P < 0.05, n = 5 for each group) (Figure  3).

VEGF and FGF2 expressed intensely on the surface of the meniscus, around the injured site and in the red zone in the miR-210 group compared with the control group.

Type 2 collagen expression was observed around the injured site of the meniscus in the miR-210 group compared with the control group.

Real-time quantitative PCR was carried out using TaqMan Gene Expression Assay probes for rno-miR-210, snoRNA-135, VEGF, collagen type 1 alpha 1 (Col1a1), FGF2 and ACTB.

Moreover, miR-210 could directly promote collagen synthesis in inner region meniscus cells.

In immunohistochemistry, VEGF and FGF2 intensely expressed on the surface of the meniscus, around the injured site and in the red zone in the miR-210 group compared with the control group (Figure  4A).

The cells were transfected with miR-210 (20 nM) or silencer negative control RNA (20 nM; Life Technologies) using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies).

Preparation of double-stranded miR-210 and small interfering RNA–atelocollagen complex.

The results of this study suggest that administration of synthetic miR-210 would be a meniscus treatment option, although the exact process and molecular mechanism of meniscal healing have not been fully elucidated.

In vitro, the inner meniscus and synovial cells were isolated from rat knee joint, and were transfected with ds miR-210 or control dsRNA.

Newly formed vessels were observed around the injured site in the miR-210 group, while little blood vessels were observed in the control.

At 12 weeks, the intra-articular injection of miR-210 had healed the injured site of the meniscus and had prevented articular cartilage degeneration.

As for the cell proliferation, miR-210 could promote cell proliferation in the meniscus.

miR-210 is recognized to have the potent function of angiogenesis, and in this study intra-articular injection of ds miR-210 could enhance the angiogenesis around the injured site of the meniscus.

In the miR-210 group, many proliferative cells were observed around the injured site.

Histological findings for six out of six rats in the miR-210 group but only for two out of six rats in the control group showed complete meniscal healing.

An intra-articular injection of double-stranded (ds) miR-210 (for control group using control dsRNA) with atelocollagen was administered immediately after injury.

Shoji and colleagues demonstrated that intra-articular injection of ds miR-210 can promote the healing of partially torn anterior cruciate ligaments through enhancement of angiogenesis [30].

The ds miR-210 and atelocollagen complex was prepared by mixing an equal volume of atelocollagen (in phosphate-buffered saline, pH 7.4) and dsRNA solution (20 μg/15 μl) and mixing by rotation at 4°C for 20 minutes.

The injured site of the miR-210 group was filled with repaired tissue stained with safranin O, while that of the control group was not repaired.

Several reports link miR-210 to the pathogenesis of cancer, so further investigation including the validation of target genes is required.

For PCR analysis, a 2 mm × 2 mm piece of meniscus including the injured site was resected from the control and miR-210 groups.

Number of blood vessels in the miR-210 group was significantly higher than that in the control group (* P < 0.05, n = 5 for each group).

Arrows, FAM-labeled ds miR-210/atelocollagen complex.

The meniscal healing score of the miR-210 group was significantly higher than that of the control group (P < 0.05, n = 6 for each group) (Figure  2A).

Therefore, we hypothesized that intra-articular injection of synthetic miR-210 could enhance the injured meniscal healing process.

Macroscopic findings showed that the injured site in the miR-210 group was morphologically healed, whereas the tear in the meniscus still remained in the control group at 12 weeks after the intra-articular injection.

In the miR-210 group, newly formed vessels were observed around the injured site, while few blood vessels were observed in the control group.

Intra-articular injection of miR-210 can accelerate meniscal healing.

In the miR-210 group, one-half of the rats showed complete meniscal healing, and the remaining rats showed high or intermediate healing.

Several papers have reported the involvement of miR-210 in angiogenesis.

ds miR-210 or ds negative control RNA were transfected into the meniscus or synovial cells using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA USA) according to the manufacturer’s protocol.

Twenty-four hours after the injection, FAM (Fluorescein amidite) labeled miR-210 was observed in the cells around the injured site.

Proliferative cells in the miR-210 group were significantly higher than that in the control group (* P < 0.05, N. S. (no significant difference), n = 5 for each group).

In our current study, the production of collagen type 2 was enhanced by the administration of miR-210 in vivo, and notably this phenomenon could be observed in isolated cultured meniscus cells.

Several groups have reported recently that miR-210 is a key player in angiogenesis in response hypoxia [25, 26].

Fluorescein amidite-labeled ds miR-210/atelocollagen complex was taken in cells of the whole meniscus after intra-articular injection, and was observed in the cells around the injured site in particular (Figure  1D).

Intra-articular injection of miR-210 prevents articular cartilage degeneration.

Intra-articular injection of miR-210 prevented articular cartilage degeneration.

In the miR-210 group, many proliferative cells that had an immunoreactivity of Ki67 were observed around the injured site.

Shoji and colleagues demonstrated that intra-articular injection of synthetic miR-210 could accelerate anterior cruciate ligament healing in the rat mo del via enhancement angiogenesis [30].

Degenerative change of cartilage in the control group was more advanced than in the miR-210 group.

Safranin O staining revealed that the degeneration of articular cartilage in the miR-210 group was lower than that of the control group.

The histological score of the miR-210 group (2.3 ± 0.8 points) was significantly higher than that of the control group (1.0 ± 1.0 points) (P < 0.05, n = 6 for each group) (Figure  1A,B,C).

Proliferative cells in the miR-210 group were significantly higher than those in the control group (P < 0.05, n = 5 for each group) (Figure  4D).

The number of blood vessels in the miR-210 group was significantly higher than that in the control group (P < 0.05, n = 5 for each group) (Figure  4C).

For fluorescence microscopy of fluorescein amidite-labeled miRNA-210 in meniscus samples, 6 μm serial sections were mounted on saline-coated glass slides, air dried and fixed with 4.0% paraformaldehyde at 4°C for 5 minutes.

We used ds miR-210 that was designed for intra-articular injection in the experimental group (sequences 66-CUG-UGC-GUG-UGA-CAG-CGG-CUG-A-87 and 87-AGC-CCC-UGC-CCA-CCG-CAC-ACU-G-66 labeled with fluorescein amidite; B-Bridge International, Mountain View, CA, USA).

Four weeks after the injection, the injured site of the miR-210 group was filled with repaired tissue while that of the control was not repaired.

4

Then we found miR-210 targeted 3′-UTR of DR6 to inhibit its expression.

The results demonstrated that miR-210 alleviated inflammation in articular cavity in OA rats by targeting DR6 and inhibiting NF-κB signaling pathway, suggesting that miR-210 might be a medical target for the treatment of OA.

Expression of IκBα and p65 in OA rats were changed compared with sham surgery group (P < 0.05), while the effect could be inhibited by miR-210 overexpression (Fig. 5e,f).

However, miR-210 overexpression inhibited the production of cytokines in SF samples (Fig. 5b–d).

In addition, cell apoptosis caused by LPS was attenuated by PDTC, DR6 siRNA and miR-210 mimic (Fig. 4b), suggesting that miR-210 targeted DR6 and protected chondrocytes from LPS by inhibiting activation of NF-κB signaling pathway.

Overexpression of miR-210 in epithelial ovarian cancer promotes tumor growth via inhibiting cell apoptosis 26.

As shown in Fig. 4, miR-210 played a similar role with PDTC to inhibit NF-κB activation by reducing the p65 expression and increasing the IκBα level in LPS treated cells.

However, a study demonstrated that overexpression of miR-210 under hypoxia was regulated by NF-κB transcriptional factor p50 23.

MiR-210 overexpression inhibits TLR4 -induced secretion of pro-inflammatory cytokines IL-6 and TNF-α 13.

MiR-210 inhibited expression of pro-inflammatory cytokines and NF-κB pathway in OA rats.

MiR-210 expression was inhibited by LPS in chondrocytes.

The regulation of p65 and IκBα caused by LPS was also inhibited by miR-210 mimic and DR6 siRNA.

How to cite this article: Zhang, D. et al. MiR-210 inhibits NF-κB signaling pathway by targeting DR6 in osteoarthritis.

The results indicated that DR6 was the direct target of miR-210.

MiR-210 is always overexpressed in tumors and inhibits cell apoptosis to promote cancer progress.

NC, normal control group; SS, sham surgery group; OA, OA mo del group; OA+miR-210, OA mo del with the treatment of miR-210 -expressing lentivirus.

The articular cartilages of medial tibial plateau were collected and stored at −80 °C for further expression analysis of miR-210, DR6, p65 and IκBα.

In conclusion, the results demonstrate miR-210 expression was decreased both in LPS -induced chondrocytes and OA rats.

As shown in Fig. 3, DR6 which is an important protein to induce cell apoptosis and activation of NF-κB was confirmed to be the target of miR-210.

DR6 was finally selected as the most likely target of miR-210 during inflammation (Fig. 3c).

Besides, transfection of siRNAs of NF-κB also reduces miR-210 expression 27.

The isolated chondrocytes were treated with LPS to induce inflammation in vitro, the expression level of miR-210 was detected by RT-PCR.

Twenty rats were randomly divided into four groups: normal control group (NC; n = 5), sham surgery group (SS; n = 5), OA mo del group (OA; n = 5), and OA mo del with the treatment of miR-210 -expressing lentivirus (OA+miR-210; n = 5).

Expression of miR-210 in chondrocytes was significantly enhanced after transfection with miR-210 mimic (Fig. 1b).

As shown in Fig. 3a,b, protein level of DR6 was increased by LPS and inhibited by miR-210 mimic.

showed that the miR-210 expression in articular cartilage of OA rats and LPS-simulated chondrocytes was much less abundant than that of normal rats, suggesting miR-210 may play an important role in OA.

MiR-210 -expressing lentivirus (Hanheng Bio-Tech Co.

The results indicate that miR-210 acts as a feedback regulator of NF-κB pathway.

MiR-210 targeted DR6 in chondrocytes.

MiR-210 mimic and DR6 siRNA inhibit the activation of NF-κB and cell apoptosis of chondrocytes.

MiR-210 protected chondrocytes through inhibiting NF-κB pathway.

MiR-210 -expressing lentivirus was injected into articular cavity of the OA rats.

MiR-210 overexpression exhibited anti-apoptotic and anti-inflammatory effects.

To confirm whether miR-210 was targeted at the 3′-UTR of DR6, a relative luciferase activity assay was performed.

To investigate the molecular mechanism of miR-210, the potential targets of miR-210 was predicted from RegRNA website.

A mutated 3′-UTR of DR6 was introduced into the potential miR-210 binding site by the two-step PCR approach.

Effect of miR-210 in OA rats.

To further evaluate the molecular mechanism of miR210, its target gene was confirmed.

Pro-inflammatory cytokines production, cell viability reduction and cell apoptosis induced by LPS were alleviated by miR-210 mimic.

In this study, transfection of miR-210 mimic exhibited anti-inflammatory and anti-apoptotic effects in LPS -induced chondrocytes.

The results also showed that miR-210 possess anti-inflammatory effects.

It has been shown that miR-210 is also associated with hypoxia pathway and protect cells from hypoxia -induced apoptosis 25.

mRNA levels of miR-210 in chondrocytes.

To investigate whether NF-κB signaling pathway was involved in the protective effect of miR-210, pyrrolidine dithiocarbamic acid (PDTC) which is an inhibitor of NF-κB was used in the study.

The results indicated that miR-210 protect the chondrocytes from LPS induced injury.

The levels of IL-1β and TNF-α were decreased in miR-210 mimic transfected cells (Fig. 2a).

As shown in Fig. 2b, the relative cell viability was decreased by 55% after the treatment of LPS, whereas the cell viability was increased by 32% after miR-210 transfection.

Previous studies indicated that miR-210 may be associated with OA 12 23.

MiR-210 was also confirmed to be a feedback negative regulator for LPS -induced inflammation 13.

Effect of miR-210 on LPS -induced chondrocytes.

As shown in Fig. 1a, the miR-210 level was decreased by 75% in LPS induced cells.

As shown in Fig. 5a, miR-210 level in the articular cartilages was decreased by 0.8-fold in OA rats and increased by 5.4-fold in lentivirus infected rats.

As shown in Fig. 2c,d, the percentage of cell apoptosis was increased to 39.1% in LPS -induced cells (P < 0.01), and reduced to 25.6% after miR-210 mimic transfection (P < 0.05).

, Shang hai, China) was injected into articular cavity of the rats in OA+miR-210 group.

The relative luciferase activity significantly decreased when the cells were transfected with the wide type of DR6 3′-UTR and miR-210 mimic (Fig. 3d).

The reporter vectors containing the wide type or mutant of DR6 3′-UTR and miR-210 mimic were cotransfected into 293T cells (ATCC, Manassas, VA, USA).

5

2.3. miR-210 Downregulates the Expression of Junction Proteins Occludin and β-Catenin.

Of critical importance, the finding that the inhibition of miR-210 with miR-210-LNA significantly reduced BBB damage and cerebral edema formation following HI insult provides a proof of concept for a novel target of potential therapeutic intervention in the treatment of neonatal HI brain injury.

Of importance, inhibition of miR-210 with miR-210-LNA significantly reduced cerebral edema, BBB leakage, and junction protein damage following HI insult, suggesting a novel mechanism of the neuroprotective effect of miR-210 inhibition in the neonatal HIE.

Moreover, our previous [33] and present studies showed that miR-210 mimic pretreatment significantly reduced expressions of target genes 48 h after injection.

Of importance, the inhibition of miR-210 with complementary locked nucleic acid (LNA) oligonucleotides 4 h after brain HI insult increases the abundance of junction proteins in the brain, and significantly reduces BBB leakage and cerebral edema, suggesting a novel mechanism of the neuroprotective effect mediated by miR-210 inhibition on neonatal HIE.

Herein, we present evidence of a novel mechanism that miR-210 impairs the BBB integrity by suppressing the expression of junction proteins, thus resulting in increased susceptibility of the BBB to HI insult in neonatal rats.

Our previous study demonstrated that miR-210 increased the vulnerability of the neonatal brain to HI insult, and miR-210 antagomir LNA treatment provided both short-term and long-term neuroprotection by the downregulation of brain miR-210 levels [33].

We reported that brain miR-210 levels were significantly upregulated as early as 3 h after neonatal HI [33].

We then determined whether miR-210 is detrimental to BBB integrity by negatively regulating the expression of junction proteins.

Our data showed that both occludin and β-catenin protein levels were significantly reduced 48 h after miR-210 injection into the brain, and inhibition of miR-210 protected occludin and β-catenin proteins from neonatal HI, suggesting that the effect of miR-210 on the increased vulnerability of the BBB in response to neonatal HI is mediated by negative regulation of junction proteins occludin and β-catenin.

Our data showed that the protein abundance of occludin and β-catenin was significantly downregulated 24 h after neonatal HI, which was reversed by the miR-210-LNA treatment but not by its negative control (Figure 4).

miR-210 mimic may produce a potential delayed effect on the neonatal brain by targeting junction proteins.

Moreover, we also found that miR-210 inhibition reduced IgG extravasation after neonatal HI.

To determine the effect of miR-210 inhibition on the BBB permeability after neonatal HI treatment, either miR-210-LNA or its negative control was delivered into the ipsilateral hemisphere of rat pups through i. c. v. injection 4 h after HI.

2.2. miR-210 Inhibition Reduces Cerebral Edema and Serum IgG Leakage Following Neonatal HI.

Therefore, to understand the mechanisms of miR-210 on BBB integrity, we focused on the expression of junction proteins.

Future study is needed to further identify whether miR-210 directly inhibit junction proteins using in vitro luciferase reporter gene assay.

In experiment 2, the purpose of posttreatment with miR-210-LNA was to reveal the potential clinic relevance of miR-210 inhibition on neonatal HI brain injury.

Recently, we demonstrated that the HI treatment significantly increased miR-210 in the neonatal rat brain and inhibition of miR-210 provided neuroprotection in neonatal HI brain injury [33].

Glucocorticoid receptor (GR), which has been determined as a target of miR-210 in our previous study [33], may also be involved in BBB damage induced by neonatal HI.

Ma Q. Dasgupta C. Li Y. Bajwa N. M. Xiong F. Harding B. Hartman R. Zhang L. Inhibition of microRNA-210 provides neuroprotection in hypoxic-ischemic brain injury in neonatal ratsNeurobiol.

To identify potential target genes of miR-210, we analyzed the seed sequence of miR-210 (UGCGUGUC) and found two candidates, OCLN and CTNNB1, encoding tight junction (TJ) protein occludin and adherens junction (AJ) protein β-catenin, respectively, and both proteins are tightly associated with maintenance of the BBB integrity [14, 36].

The transcripts of both genes contained potential miR-210 seed targeting sequences in their 3′ UTRs, as shown in Figure 3A.

The present study demonstrated that miR-210 was detrimental to the BBB permeability and cerebral edema formation after neonatal HI insult in rats, which was mediated by the negative regulation of BBB junction proteins occludin and β-catenin.

However, it remains undetermined whether and to what extent miR-210 participates in the regulation of the BBB disruption in neonatal HI brain injury.

Fasanaro P. D’Alessandra Y. di Stefano V. Melchionna R. Romani S. Pompilio G. Capogrossi M. C. Martelli F. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3J.

To further investigate the role of endogenous miR-210 on the regulation of BBB junction proteins, we then tested whether miR-210 inhibition protected junction proteins occludin and β-catenin from neonatal HI insults.

For instance, miR-210 plays important roles in the regulation of endothelial cell survival [30, 31] and vascular angiogenesis in response to hypoxia [32].

Zeng L. He X. Wang Y. Tang Y. Zheng C. Cai H. Liu J. Wang Y. Fu Y. Yang G. Y. MicroRNA-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brainGene Ther.

Given the lack of effective therapy strategies against neonatal HIE, the present study deepens our understanding of the neuroprotective effect of miR-210-LNA and will be beneficial for the development of treatment strategies.

In conclusion, the main finding of this study is that miR-210 plays a detrimental role in BBB integrity by its negative regulation of junction proteins, thereby contributing to cerebral edema formation after neonatal HI.

Therefore, in addition to junction proteins, miR-210 may orchestrate the BBB integrity by regulating other signaling pathways in response to neonatal HI.

Moreover, miR-210-LNA but not its negative control also significantly reduced the level of serum IgG in the brain tissue induced by HI insult (Figure 2B).

In experimental protocol #2, pups were divided into four groups: (1) Sham, (2) HI, (3) HI + miR-210-LNA (50 pmol), and (4) HI + miR-210-LNA negative control (Neg.

To exclude the possibility that HI -induced brain injury concealed the detrimental effect of miR-210 to the neonatal brain, we conducted pretreatment with miR-210 mimic and detected changes of the vulnerability of neonatal brain to HI insult.

Our data showed that miR-210 mimic significantly increased cerebral edema formation after neonatal HI, while miR-210-LNA significantly reduced cerebral edema caused by HI insult.

Previous studies showed that miR-210 modulated endothelial cell response to hypoxia in vitro [30, 31] and angiogenesis after ischemic stroke in animals [32].

miR-210 mimic (Qiagen, Valencia, CA, USA), miR-210-LNA (Exiqon, Woburn, MA, USA), and their negative controls were prepared according to the manufacturers’ instructions, and were administered into the ipsilateral hemisphere of rat pups with a total volume of 2 µL via i. c. v. injection (coordinates: 2 mm posterior, 1.5 mm lateral, 3 mm below the skull surface) as we described previously [33].

By comparison of the seed sequence of rat miR-210 with the 3′ UTRs of the transcripts of junction proteins, we found that the seed sequences of miR-210 are complementary to the 3′ UTRs of the transcripts of OCLN and CTNNB1, encoding tight junction protein occludin and adherens junction protein β-catenin.

2.1. miR-210 Mimic Exacerbates Cerebral Edema and Immunoglobulin G (IgG) Leakage Following Neonatal HI Insult.

The dose of miR-210 mimic or miR-210-LNA was determined in our previous study [33] whereby miR-210 increased the vulnerability of neonatal brain to HI insult, and miR-210-LNA provided neuroprotective effect with those doses.

This finding uncovers a new mechanism for the miR-210-LNA mediated neuroprotective effect on neonatal HI brain injury, which has been determined in our previous study [33].

To explore whether miR-210 increased the vulnerability of the BBB in neonatal brain to HI insults, either miR-210 mimic or its negative control was delivered into the brain of rat pups through intracerebroventricular (i. c. v. ) injection.

2.4. miR-210-LNA Treatment Protects Junction Proteins Occludin and β-Catenin from Neonatal HI Insult.

However, the mechanisms underpinning the detrimental effect of miR-210 in the neonatal brain remain unclear.

These findings implicate that miR-210 may play a critical role in BBB damage following neonatal HI insult.

We found that miR-210 exacerbated the vulnerability of the BBB in neonates to HI insult by increasing serum IgG extravasation into brain tissue and cerebral edema.

Moreover, the effect of miR-210 on BBB integrity will also need to be determined in an in vitro BBB mo del using brain microvascular endothelial cells.

Thus, the neuroprotection of miR-210-LNA may be attributed, in part, to its effect on the preservation of BBB integrity and the reduction of cerebral edema.

Our results showed that miR-210-LNA treatment significantly reduced cerebral edema by about 50% at 48 h after HI (Figure 2A).

Based on this point, the reduction of brain edema by miR-210-LNA treatment not only diminished brain edema but also reduces other consequences resulting from brain edema such as cell death.

Immunofluorescence staining showed that IgG leakage observed in microvessels in the cortex neighboring the core infarct region (perilesional area), identified by cell loss and pyknotic nucleus, was reduced by miR-210-LNA at 24 h after HI (Figure 2C,D).

In experimental protocol #1, pups were divided into two groups: (1) miR-210 mimic (60 pmol), and (2) miR-210 negative control (Neg.

Moreover, in the present study, we revealed that miR-210 mimic was detrimental to BBB integrity in acute phase after HI.

Thus, in the present study, we administered miR-210 mimic into the brain of neonatal rats and assessed the changes of BBB integrity in response to HI insult.

The result showed that miR-210 mimic significantly reduced the protein levels of occludin and β-catenin 48 h after injection as compared to its negative control (Figure 3B), suggesting that the effect of miR-210 on the increased vulnerability of BBB in response to neonatal HI is mediated by negative regulation of junction proteins occludin and β-catenin.

6

The present study has demonstrated for the first time that 1) sevoflurane pretreatment can promote the therapeutic effect of BMSCs transplantation on AMI through inhibiting apoptosis, enhancing their paracrine function and activating resident CSCs; 2) sevoflurane pretreatment can inhibit hypoxia -induced BMSCs apoptosis and promote their migration and cytokines secretion through regulating miR-210 and its target genes Casp8ap2 and PTPN2.

Caspase-8 -associated protein 2 (Casp8ap2) and protein tyrosine phosphatase, non-receptor type 2 (PTPN2) have been confirmed as target genes of miR-210 [27, 28], whose expression is decreased in [SFpre]BMSCs under a hypoxic condition and increased after exposure to anti-miR-210, indicating that sevoflurane regulates Casp8ap2 and PTPN2 by miR-210.

Base on the evidence that miR-210 inhibited hypoxia -induced apoptosis of stem cells by down-regulation of Casp8ap2 [28], it is speculated that sevoflurane may alleviate the hypoxia -induced apoptosis of BMSCs via miR-210/ Casp8ap2 pathway.

As shown by in vitro results of RT-PCR and Figure 4A, the expression of miR-210 was up-regulated in BMSCs exposed to hypoxia and [SFpre]BMSCs (P < 0.05, Figure 4B).

To explore the underlying mechanism, in vitro experiments were carried out to assess the potential of [SFpre]BMSCs against hypoxia by determining the expression of miR-210 (an important anti-hypoxic factor [18, 19]) and its target gene caspase 8 associated protein 2 (Casp8ap2) and protein tyrosine phosphatase, non-receptor type 2 (PTPN2), cell migration, apoptosis and secretion of cytokines, as well as the induced activation of cardiac stem cells (CSCs).

Compared with untreated BMSCs, promoted migration, inhibited apoptosis, increased cytokine secretion, and enhanced activation to CSCs were detected in [SFpre]BMSCs exposed to profound hypoxia and serum deprivation, via up -regulating miR-210 expression (P < 0.05).

Sevoflurane influenced the expression of miR-210 and its target gene.

Figure 4The in vitro expression of miR-210, Casp8ap2 and PTPN2 in BMSCsRepresentative photographs of miR-210 expression and the ratio to U6 are shown in (A and B) respectively.

As an critical regulator of cell anti-hypoxia [18, 27], miR-210 has been identified to contribute to protective mechanisms of SFpre by our in vitro experiments, although other mechanisms may also be involved in such process, including regulation of mitochondrial respiratory function, attenuation of oxidative stress and inhibition of Beclin 1 -mediated autophagic cell death [9– 11].

To knock down endogenous miR-210, miR-210 inhibitor was transfected to BMSCs with Lipofectamine 2000 (Invitrogen Corporation, CA, USA), followed by a 48 hour incubation.

Our in vivo and in vitro studies revealed that pretreatment by sevoflurane can promoted the secretion of VEGF, bFGF and SDF-1α by BMSCs under a hypoxic condition, and that such promotion can be attenuated by anti-miR-210, indicating that sevoflurane may regulate BMSCs paracrine function through miR-210 and its target gene PTPN2, although the larger number of BMSCs protected by sevoflurane may also account for increased cytokine secretion.

As shown by the present study, anti-miR-210 could significantly inhibit SFpre -induced BMSCs activation, which indicates that miR-210 is involved in sevoflurane regulation of BMSCs migration and paracrine besides apoptosis.

Compared with BMSCs, the expression of miR-210 target gene Casp8ap2 and PTPN2 was decreased in [SFpre]BMSCs under a hypoxic condition (P < 0.001, Figure 4C and 4D), which could be reversed by anti-miR-210.

Sevoflurane preconditioning can protect BMSCs against hypoxia by activating miR-210 expression and promote their paracrine functions and effects on resident CSCs.

Representative photographs of miR-210 expression and the ratio to U6 are shown in (A and B) respectively.

In vitro experiments were performed to determine the expression of miR-210 in BMSCs exposed to sevoflurane and the effect of sevoflurane on BMSCs’ migration, apoptosis and secretion of cytokines under hypoxic condition, as well as cytokine -induced CSCs activation.

The in vitro expression of miR-210, Casp8ap2 and PTPN2 in BMSCs.

As described previously, the expression of miR-210 was determined by RT-PCR with U6 as the internal control.

More importantly, this increase was more obvious in [SFpre]BMSCs compared with the BMSCs (P < 0.05), suggesting that sevoflurane may enhance the expression of miR-210 under a hypoxic condition.

Thereafter, the expression of miR-210, Casp8ap2 and PTPN2, cell migration and apoptosis were observed, and the supernatant was harvested for analyzing the cytokine secretion and evaluating their effects on CSCs.

Additionally, miR-210 may be involved in this protective effect of sevoflurane.

anti-miR-210.

Therefore, miR-210/ PTPN2 pathway may contribute to sevoflurane -induced BMSCs migration under hypoxic condition.

Sevoflurane improved migration, apoptosis and cytokines secretion of BMSCs by miR-210.

These changes could be reversed by anti-miR-210 (P < 0.05), indicating that miR-210 is involved in the activation of BMSCs induced by sevoflurane.

7

Western blot analysis demonstrated that caspase-3, caspase-9 and bax protein levels decreased and bcl-2 expression increased in miR-210 overexpression rats, suggesting that miR-210 suppressed neuronal apoptosis by inhibiting caspase activity and regulating the balance between bcl-2 and bax levels.

Although miR-210 was reported to be upregulated in both normal and transformed hypoxic cells [3, 6, 12], mouse mo dels of brain transient focal ischaemia [4] and cardiac hypertrophy/cardiac failure, and in placentas from patients with preeclampsia, our data showed that miR-210 expression was downregulated in the brain 72 h after HI injury than in normal control (Figure 1).

MiR-210 was robustly downregulated in the brain of rats intraventricular injected with miR-210 inhibitor, while upregulated in the brain of rats intraventricular injected with miR-210 minic, which confirmed that animal mo dels were prepared successfully (Figure 1).

So, the level of miR-210, which is robustly upregulated by HIF-1 α, is dependent on the duration after HI injury.

Recent study suggested that miR-210 was expressed in both brain and blood of rat middle cerebral artery occlusion (MCAO) mo del [4, 5].

Reagents including miR-210 minic (2.5 mg/kg), miR-210 inhibitor (2.5 mg/kg), or physiological saline (2.5 mg/kg) were injected with a microosmotic pump (Alzet 1007D; Durect Corp, Cupertino, CA, USA) into the lesioned side (left side) of the lateral ventricle (coordinates: anterior/posterior −0.9 mm, left 1.5 mm relative to bregma, and dorsoventral −5.0 mm from the dural surface).

There were few TUNEL -positive cells in miR-210 overexpression rats.

These pieces of evidence suggested that miR-210 has a neuroprotection and restoration feature by suppressing neuronal apoptosis.

We confirmed the expression level of miR-210 using quantitative real-time PCR analysis.

We next assessed the effects of miR-210 on apoptosis-related protein expression.

In particular, microRNA-210 (miR-210), which is activated by HIF-1 α for the hypoxic induction [1], is a unique miR that is evolutionarily conserved and ubiquitously expressed in hypoxic cell and tissue types [2].

Furthermore, expression of miR-210 in human umbilical vein endothelial cells (HUVEC) results in increased tubulogenesis and increased vascular endothelial growth factor (VEGF) -induced cell migration through the repression of the receptor tyrosine kinase ligand Ephrin-A3 [6].

Recently, Hu et al. demonstrated that delivery of miR-210 through a nonviral minicircle vector in the ischemic heart can improve heart function by promoting angiogenesis and inhibiting apoptosis [10].

Also, Liu et al. reported that miR-210 was downregulated in both brain and blood 24 h after brain ischemia compared with untouched control animals [5].

Effects of MiR-210 on Neuronal Apoptosis and Protein Expressions of Caspase-3, Caspase-9, Bax, and bcl-2. 4. Conclusions.

A total RNA (10 ng) was reverse transcripted to cDNA with 1 mM dNTPs (with dTTP), 50 U reverse transcriptase 1  μL, 4 U RNase inhibitor in the presence of specific miR-210 or U6 stem loop reverse transcriptase primers in a 15  μL system buffered by RT Buffer and DEPC water; following the thermal cycle program of 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min, cDNA was stored at −20°C.

MiR-210 Expression.

Western blot analysis demonstrated that caspase-3, caspase-9, and bax protein levels were enhanced in miR-210 block rats and decreased in miR-210 overexpression rats compared to controls.

While miR-210 was initially thought to be intergenic, a more recent study showed that it is in fact contained within the sequence of a transcript with virtually unknown own function (AK123483) [3], which is also hypoxia inducible.

The lower miR-210 levels at 72 h after HI injury suggested that the repair function of miR-210 was diminished at late stage after neonatal HI injury.

So, we hypothesized that miR-210 may have essential functions in HIE despite its role during HIE is not understood.

So, miR-210 delivery through circulation may work as a novel therapeutic intervention for treatment of HIE.

Therefore, we hypothesized that miR-210 may lead to a novel therapy for HIE and showed the relationship between miR-210 and HIE in this study.

Additional investigations are needed to demonstrate the differential expression of miR-210 in the brain in different time points after HI injury.

Finally, the relative miR-210 level was normalized to the endogenous control U6 expression for each sample in triplicate and was calculated by the 2 [−ΔCt] method.

Likewise, our data also revealed that miR-210 in the presence of hypoxia can prevent cell apoptosis.

By contrast, the ratio of TUNEL -positive cells increased obviously in miR-210 block rats (Figure 2).

The animal data and cell culture results suggested that miR-210 was elevated immediately in response to hypoxia and subsequently decreased several days later [6, 13].

Previous studies have shown that miR-210 can protect cells from hypoxia -induced apoptosis [6, 10, 16].

MiR-210 plays multiple crucial roles in the cellular regulation in response to low oxygen including ischemic brain injury.

Conversely, miR-210 blockade in the presence of hypoxia induces apoptosis [6].

8

The miR-210 upregulation seems to be dependent on the exercise performing because the group that performed voluntary exercise showed a stronger miR-210 expression than crocin group.

As shown in Figure 5, following crocin administration and exercise performing, the heart expression level of miR-210 was significantly upregulated in Cro and Exe groups when compared to control group (p < 0.01 and p < 0.001, respectively).

[26] Previous research has indicated that miR-210 may improve angiogenesis through the negative regulation of its target gene, ephrin A3, which is an important member of the ephrin angiogenesis regulatory gene family.

[25] When miR-210 is overexpressed in endothelial cells, the ability of these cells to form blood vessels becomes pronounced, more than that of cells with normal levels of expression.

[20] MiR-210 induction is a virtually constant feature of the hypoxic response that is important in the pathogenesis of several human diseases, such as heart disease.

MiR-210, a hypoxia-specific miRNA, depends on HIF activation and upregulated after hypoxia.

Confirming miR-210 proangiogenic role, up-regulation of miR-210 in CD34+ cells increased tissue perfusion and capillary density in a mouse mo del of hind limb ischemia.

Crocin and voluntary exercise improve heart angiogenesis possibly through enhancement of miR-126 and miR-210 expression.

The present study was undertaken to clarify the effect of crocin and voluntary exercise on miR-126 and miR-210 expression in cardiac myocytes of diabetic rats.

[24] Expression of miR-126 and miR-210 was assessed by qRT-PCR.

30, 31 These data are in line with the observations made by Anja Bye et al. [32] regarding significant increase in miR-210 expression in subjects with low Vo2max following the exercise activity.

This study shows that crocin in combination with voluntary exercise promotes cardiac angiogenesis and this may be related to expression of miRNA-126 and miR-210.

In the present study, we demonstrated that miR-126 and miR-210 expression of rat cardiac tissue increased in crocin, voluntary exercise, and exercise-crocin groups.

Effects of crocin combined with voluntary exercise on miR-210 expression in the heart tissue.

Figure 5Effect of crocin and voluntary exercise on miR-210 expression levels.

Furthermore, our findings showed that crocin administration and voluntary exercise performing increased the expression of heart miR-210.

[9] In the current study, we observed that crocin and voluntary exercise increased miR-210 expression levels in heart tissue using quantitative real-time PCR analysis.

A probable mechanism is that during exercise, local hypoxic conditions in the cardiac muscle can occur and hypoxic situation trigger a number of physiological responses such as angiogenesis through HIF-1α -induced miR-210 expression.

In addition, combination of exercise and crocin amplified their effect on miR-126 and miR-210 expression, and angiogenesis.

MiR-126, miR-210 expression and CD31 in the heart increased in both crocin and voluntary exercise groups compared with control group.

18, 19 MiR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary like structures as well as VEGF -driven cell migration.

On the other hand, the expression of miR-210 increased significantly in Cro-Exe group compared with control group (p < 0.001).

Akt, ERK1/2 protein levels, miR-126 and miR-210 expression were measured in the heart tissue.

The aim of this study was to evaluate the effect of crocin and voluntary exercise on miR-126 and miR-210 expression levels and angiogenesis in the heart tissue.

The 2 [-(ΔΔCt)] method was used to determine relative quantitative levels of miR-126 and miR-210.

For Akt, ERK, CD31, miR-126, and miR-210 parameters, data were analyzed using two-way ANOVA followed by Tukey's post hoc test.

[28] Preclinical work demonstrated that intracardiac injections with a minicircle vector carrying miR-210 in a mouse mo del of myocardial infarction promoted significant improvement of left ventricular fractional shortening, decreased cellular apoptosis, and increased neovascularization.

9

MiR-137 showed a trend of upregulation after 4 h post-OGD and continued to be upregulated as well at 6, 8 and 12 h. MiR-210 was upregulated after 6 h by >1.6-fold and significantly upregulated at 8, 12 and 24 h (p<0.05) suggesting a strong involvement of this miRNA in astrocytic response to ischemia (Figure 1).

MiR-21 is significantly upregulated at 12 h and 24 h in astrocytes however in neurons upregulation occurred only after 24 h. MiR-210 is significantly upregulated in astrocytes at 8 h, 12 h and 24 h. In neurons, miR-210 showed a significant variability between each set of experiments.

They found that miR-21 and miR-210 expression are upregulated at 24 h after reperfusion that is in line with our findings in neurons and astrocytes exposed to OGD conditions.

MiR-210 showed a trend of upregulation after 6 h and a significant upregulation after 8 h in astrocytes (Figure 1).

Differently from Jeyasselan [32] study in transient ischemia mo del and our study in cell cultures subjected to OGD conditions, Liu [34] found a 1.6 fold downregulation of miR-210 in the hippocampus of a transient ischemia of MCAo rat mo del.

In our study, miR-210 is significantly upregulated in astrocytes after 8 h. While our efforts in neurons are focused on finding those miRNAs involved in apoptosis after ischemic insult; in astrocytes it is important to study those miRNAs that may control their function in protecting the CNS environment after ischemia.

Hypoxia has been shown to induce the upregulation of miR-210 in cancer and non-cancer cells [7], [12].

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.

MiR-210 was more than 2-fold upregulated at 12 h and 24 h (Figure 1).

Zhang and colleagues [72] have recently described that miR-210 influences the hypoxic response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

This might suggest that miR-210 is tightly controlled in ischemic conditions in neurons and small changes in experimental conditions may affect its expression significantly.

IGF-I did not have an effect on the expression levels of mir-21, mir-30b, mir-107, mir-137, or mir-210 in neurons.

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

Unfortunately, no definitive conclusions could be drawn for miR-210 involvement in neurons exposed to OGD.

10

Our data showed that miR-210 expression was up-regulated after 15 min ischemia and 6 h reperfusion, and the elevated miR-210 expression peaked at 96 h post-injury.

In particular, HRS beneficial effects against I/R injury were likely exerted by Treg, TGF-β1 up-regulation and miR-21, miR-210, NF-κB and TNF-α down-regulation.

FOXP3 expression is negatively regulated by miR-210 direct binding to their two target sites in its 3-UTR [48].

Some study found that miR-210 was down-regulated in Tregs compared with controls and it has potential target sites in the 3-UTR of FOXP3.

Compared with the sham and sham + H [2] groups, miR-210 and miR-21 expression in group I/R was significantly increased at 24 and 96 h after reperfusion (P < 0.05), while miR-210 and miR-21 expression in group H [2] was significantly decreased at 24 and 96 h compared with group I/R (P < 0.05) (Fig.   2).

The trend of miR-21 expression was similar to that of miR-210.

The expression of miR-210 and miR-21 in all groups was negatively correlated with the pyramidal cells number in the CA [1] area of the hippocampus at 24 h after reperfusion (P < 0.05, Pearson correlation coefficient r = −0.65 and P < 0.05, r = −0.84, respectively).

Values are mean ± SD, n = 4. b miR-210 expression by qRT-PCR analysis in the hippocampus at the indicated times after injury.

HRS Modulated miR-210 and miR-21 Expression.

When the cerebral I/R injury was treated with HRS immediately after reperfusion, miR-210 and miR-21 expressions were decreased significantly at 24 and 96 h. Our results of an increased miR-210 and miR-21 after I/R injury and their decrease in the HRS treated rats, together with the histological examination and behavioral test results shown in our study, suggested that miR-210 and miR-21 may be considered markers of cerebral I/R injury.

Fig. 2MicroRNA-21 and MicroRNA-210 expression are altered in the hippocampus after I/R.

HRS Regulated miR-210 and miR-21 in the Hippocampus After I/R.

Many studies have identified miRNA-210 (miR-210) as a unique hypoxamirs that is robustly and ubiquitously induced in both transformed and primary cell types [36, 37].

Since increasing evidence suggests that miRNA expression profile in cerebral I/R injury changes significantly, miR-210 and miR-21 in the hippocampus were measured at 6, 24 and 96 h after reperfusion.

11

Angiogenic microspheres up-regulate miR-210 expression and down-regulate ephrin-A3 expression in spinal cord tissues.

At the same time, miR-210 up-regulation directly suppresses ephrin-A3 expression, stimulating neurogenesis at the injury site.

Ujigo et al. reported that miR-210 was upregulated at the injury site after spinal cord injury and promoted angiogenesis by suppressing ephrin-A3 expression 20.

Local administration of miR-210 at sites of spinal cord injury in rats knocks down ephrin-A3 expression, promoting angiogenesis 20.

Angiogenic microspheres stimulate miR-210 expression in spinal cord tissues.

Therefore we wanted to examine whether the enhanced neural regeneration observed in our animals treated with angiogenic microspheres was associated with changes in expression of miR-210 and ephrin-A3.

The miR-210 is implicated in the regulation of many cellular processes, including cell cycle, development, and membrane trafficking 39 40.

One pathway is a positive feedback loop in which angiogenesis activated by the three factors promotes miR-210 expression, which induces endogenous VEGF production, leading to further angiogenesis.

Real-time polymerase chain reaction (PCR) was performed using TaqMan microRNA assay kits (Applied Biosystems) for mature miR-210 and its target gene, ephrin-A3 (Efna3; XM_574979).

At 2, 4 and 8 weeks after microsphere injection, tissues from 4 rats in each group (per time point) were analyzed by ELISA and real-time PCR to measure changes in VEGF, Ang-1 and bFGF as well as in endogenous expression of miR-210 and ephrin-A3.

Each tissue block was divided into two equal parts along its longitudinal median: one part was analyzed using ELISA to measure release of angiogenic factors; the other part was analyzed using real-time PCR to detect changes in expression of miR-210 and ephrin-A3.

We observed that miR-210 levels at the lesion site were significantly higher in animals that received angiogenic microspheres than in those that received empty microspheres.

12

MiR-377 and miR-210 are the most significantly dysregulated miRs in hypoxia -treated rat MSCs compared to normoxia -treated MSCs (the green stands for down-regulation, and the red stands for upregulation).

When the signal density of miRs was cut off by a value of 100, a group of 13 miRs were significantly up-regulated including miR-210, -25, -450a, -130a, -3593-3p, -34c*, -214, -181a, -23b, -34a, -31, -31*, and -140*; whereas 20 miRs (miR-377, -146a, -222, -652*, -466b-1*, -664-1*, -196c*, -466c*, -29c, -32*, -195, -466b, -188, -146b, -92a, let-7b, -466b-2*, -466d, -485*, and -181c) were significantly down-regulated in MSCs under hypoxic conditions.

MiR-210 is a key player of cell response to hypoxia [25] and indirectly up-regulates VEGF -mediated angiogenesis by targeting Ephrin-A3 [15], and enhances MSC -mediated angiogenesis [26].

In addition, Fasanaro, et al. [15] first reported that hypoxia -driven miR-210 promotes angiogenic response in ECs by down-regulation of EFNA3, an ephrin family member involving vascular development.

Recent studies also indicate that a panel of miRNAs (i. e., miR-10, miR-15b, miR-16, miR-20a, miR-20b, miR-27a, miR-126, miR-145, miR-195, miR-205, and miR-210) is involved in the regulation of VEGF expression in ECs and tumor cells [16]– [26].

While miR-210 is well recognized to have pro-angiogenic property in vivo and in vitro [15], [17], it is unknown whether hypoxia-responsive miR-377 also regulates angiogenesis.

miR-210 was the most significantly increased miR in hypoxia -treated MSCs (Fig. 1B), a finding consistent with previous observations in tumor cells, endothelial cells, and cardiomyocytes in responsive to hypoxia [15], [30], [31].

13

Regarding changes in the expression profiles of miRNA mediated by adenoviral transduction, up-regulation of miR-2137 and miR-204-5p and down-regulation of miR-210-3p were also confirmed (Fig 6D, 6E and 6G), being the expression levels of miR-210-3p and miR-204-5p in Ad-transduced B13 cells similar to those displayed by rat islets.

Interestingly, PNM overexpression in B13 cells upregulated the expression of miR-137-3p, miR-135a-5p and miR-204-5p and repressed the expression of miR-210-3p to the same levels of those detected in rat islets, highlighting the potential role of these miRNAs in the reprogramming process.

Furthermore, expression levels of miR-137-3p, miR-135a-5p, miR-204-5p, miR-210-3p in reprogrammed B13 cells were consistent with the expression levels detected in rat islets and markedly differed from those displayed by rat exocrine tissue.

0145116.g006 Fig 6Relative miRNA expression levels of miR-200c-3p (A), miR-141-3p (B), miR-325-3p (C), miR-2137 (D), miR-210-3p (E), miR-181a-5p (F), miR-204-5p (G), miR-455-3p (H), miR-137-3p (I), miR-135a-5p (J), miR-384-5p (K) in rat exocrine fractions, rat islets, AR42J cells, B13 cells and B13 transduced with Ad-GFP or Ad-PNM at 4 days post-transduction, quantified by individual qPCR assays.

Relative miRNA expression levels of miR-200c-3p (A), miR-141-3p (B), miR-325-3p (C), miR-2137 (D), miR-210-3p (E), miR-181a-5p (F), miR-204-5p (G), miR-455-3p (H), miR-137-3p (I), miR-135a-5p (J), miR-384-5p (K) in rat exocrine fractions, rat islets, AR42J cells, B13 cells and B13 transduced with Ad-GFP or Ad-PNM at 4 days post-transduction, quantified by individual qPCR assays.

14

Importantly, not all miRNA were downregulated in the CKD VSMC as miR-210, previously shown to be upregulated in acute kidney injury [30], was upregulated in CKD VSMC compared to normal littermates.

We also analyzed miR210, a miRNA known to be upregulated in acute kidney injury [29], [30], demonstrating increased expression in the VSMC from our CKD animals (Figure 3A), confirming our findings were not due to uniform miRNA suppression.

was performed to determine the expression of miR-125b, miR-145, miR-155 and miR-210 and normalized by U6 (A).

Target-specific PCR primers (miR-125b, miR-145, miR-155 and miR-210) were obtained from Applied Biosystems.

15

As seen in Figure 3, the expression levels of miR-146a, miR-210 and miR-27a were up-regulated, while the expression levels of miR-135b and miR-33 were down-regulated (* p < 0.05).

The five differentially expressed miRNAs (miR-146a, miR-210, miR-27a, miR-135b and miR-33) in the TLE rat hippocampus as detected by the Rat miRNA microarray were confirmed using qPCR (Data are presented as the mean ± SEM, * p <0.05; n = 6/TLE rats, n = 6/control).

To validate the altered expression of miRNAs as detected by miRNA microarray, miR-146a, miR-210, miR-27a, miR-135b and miR-33 were selected for confirmation by quantitative real-time PCR.

Some of the deregulated miRNAs (miR-146a, miR-210, miR-27a, miR-135b and miR-33) were confirmed using qPCR.

16

Based on previous work, the expression of the two increased miRNA species miR-375 and miR-210 and the three decreased miRNAs, miR-203, miR-185 and miR-450a, is involved in the regulation of insulin secretion and pancreatic β cell function [11, 26, 27, 28].

The observed increase in blood might be a result of modifications in organ function or morphology as increased circulating miR-210 levels were detected, e. g., under hypoxic conditions in cardiac tissue, and miR-210 expression increases during myocardial infarction in other studies [32].

In a recent study, decreased miR-203 and miR-210 expressions were detected in pancreatic islets of young pre-diabetic and diabetic db/ db mice and in mice fed with a high fed diet [26] and were strongly associated with β cell dysfunction [20, 22].

Our study revealed a continuous elevation of miR-210 blood level in ZDF rats.

Overall, the blood level of 3 miRNA species is significantly elevated, namely miR-133a, miR-375 and miR-210.

The study of Nesca et al., 2013, observed a decrease in miR-210 in pancreatic islets [26].

At late-stage diabetes, the circulating level of 12 miRNAs was specifically altered; the circulating level of miR-375, miR-210 and miR-133a was increased, and the circulating levels of let-7i, miR-140, miR-450a, miR-185, miR-186, miR-151-3p, miR-203, miR-16 and miR-685 were strongly diminished versus their levels at the pre-diabetes stage (Figure 4).

17

For example, miR-210-3p is known as the “master” hypoxamiR; it is induced by hypoxia in a HIF -dependent manner and has been shown to exhibit cardioprotective effects, such as preservation of cardiac function, upregulation of neovascularization, and inhibition of apoptosis [18].

miR-210-3p derived from exosomes secreted by cardiac progenitor cells (CPCs) inhibits cardiomyocyte apoptosis by targeting ephrin A3 and PTP1b [14].

miR-21-5p and miR-210-3p are enriched in exosomes secreted from mouse cardiac fibroblast-derived induced pluripotent stem cells (iPS cells), and prevent H9c2 cells apoptosis by regulating Nanog and HIF-1α, respectively [13].

18

In a recent study by our lab, treatment with the microRNA-210 inhibitor (promotes up-regulation of GR expression) was given 4 h post HI-injury in neonatal rats via both direct ICV injection as well as a novel method of intranasal instillation [10].

Ma Q. Dasgupta C. Li Y. Bajwa N. M. Xiong F. Harding B. Hartman R. Zhang L. Inhibition of microRNA-210 provides neuroprotection in hypoxic-ischemic brain injury in neonatal rats Neurobiol.

19

In our study, miR-210 showed slight but statistically significant female bias in expression, with females showing 1.5 fold higher expression than males at 15 weeks of age (Table  2).

Furthermore, elevated miR-210 expression in renal carcinoma tissue is predictive of better clinical outcomes for clear cell renal cell cancer patients [65].

MiR-210, which exhibits female-biased expression (1.5 fold at 15 weeks), has been identified as a potential biomarker in plasma samples for predicting survivability in AKI patients [16].

Furthermore, three circulating plasma miRNAs (miR-16, miR-320, and miR-210) have been identified in acute kidney injury (AKI) patients as potential renal biomarkers [16].

20

Another study used lentiviruses to silence β-catenin in gastric cancer cell lines and found the dysregulated expression of a number of miRNAs including increased expression of miR-210 and decreased expression of miR-455-3p [60].

Both miR-210-5p and miR-455-3p were also dysregulated in our study, and we have previously shown that Cd alters β-catenin sub-cellular localization and activity in NRK-52E rat proximal tubule epithelial cells and causes a loss of β-catenin at cell–cell contacts [6, 53, 54, 55, 56, 57].

21

miR-210 was selected as the ‘master hypoxamiR’, since this miRNA has been shown to exhibit high and consistent upregulation under hypoxia in the majority of cell types (17).

The ‘master hypoxamiR’, miR-210, has been shown to exhibit high and consistent upregulation under hypoxic conditions in the majority of cell types (17); however, the cells used in this study were mature.

In the present study, the expression of miR-210 did not change significantly in the developing lung following intrauterine hypoxia exposure when compared with the normoxic group.

22

Furthermore, several miRs, including miR-210 (which regulates angiogenesis) [14], miR-199a (which modulates hypoxia-inducible factor-1 α (HIF-1 α) expression) [16], and miR-494 (which upregulates p-Akt, HIF-1 α, and heme oxygenase-1 expression) [17], protect cells from hypoxia- or ischemia -induced damage.

23

Recent data have shown numerous miRNA dysregulations during MI, for example, MiR-210 and miR-1 were proven to improve cardiac function following MI by enhancing angiogenesis and inhibiting cardiomyocyte apoptosis [10, 11]; MiR-150 was shown to be downregulated in patients with acute myocardial infarction (AMI), atrial fibrillation, dilated cardiomyopathy and ischemic cardiomyopathy [12– 14]; and overexpression of microRNA-99a attenuates heart remo deling and improves cardiac performance following myocardial infarction [15].

24

It has previously been reported that upregulating miR-21 or downregulating miR-181a reduces H [2]O [2] -induced apoptosis in H9C2 cardiomyocytes, and that overexpressing miR-210 contributes to the protective effect of insulin against apoptosis in H [2]O [2] -treated H9C2 cardiomyocytes [34– 36].

25

For instance, miR-210 seems to have a function as neuroprotector by inhibiting cell apoptosis, with an up-regulation in pheochromocytoma (PC12) cells compared with normoxic controls (Qiu et al., 2013).

Neuroprotective effects of microRNA-210 against oxygen-glucose deprivation through inhibition of apoptosis in PC12 cells.

26

Mutharasan RK Nagpal V Ichikawa Y Ardehali H microRNA-210 is upregulated in hypoxic cardiomyocytes through Akt- and p53 -dependent pathways and exerts cytoprotective effectsAmerican journal of physiology.

Validation of a selected few by qPCR identified 10 miRNAs - miR-133b-3p, miR-208b-3p, miR-21-5p, miR-125a-5p, miR-125b-5p, miR-126-3p, miR-210-3p, miR-29a-3p, miR-494-3p and miR-320a, that were significantly up-regulated in HF myocardium compared to normal controls.

MiR-210-3p is closely related to hypoxia 41, 42, and interestingly, both miR-320a and miR-494 are reported to be associated with cardiac apoptosis induced by ischemia 43, 44.

27

We found that up-regulated miRNAs (miR-346, miR-135b, miR-30ab, miR-344, miR-18a, miR-99a, miR-210, miR-207, miR-18a, and miR-129) in ARDS were inversely correlated with the expression of their predicted targets such as Gabrb1, Mdh1, Eif2ak1, Fbln5, and Tspan8.

28

Nevertheless the altered expression of miR-145 and miR-210 was not unique for the acute phase as we found them to be increased in recovery phase (Figure 3).

Ho A. S. Huang X. Cao H. Christman-Skieller C. Bennewith K. Le Q. T. Koong A. C. Circulating miR-210 as a novel hypoxia marker in pancreatic cancerTransl.

Zhao A. Li G. Péoc’h M. Genin C. Gigante M. Serum miR-210 as a novel biomarker for molecular diagnosis of clear cell renal cell carcinomaExp.

miR-210 has also been proposed as a biomarker for a variety of conditions such as pancreatic and renal cell cancer as well as congestive heart failure highlighting its non-specificity and inapplicability, particularly in clinical diagnosis of stroke [20– 22].

We observed that circulating miR-145 and miR-210 were indeed significantly increased in stroke patients with strong AUC values of 0.90 and 1.0 respectively.

Incidentally, miR-145 and miR-210 had been previously reported to be potential biomarkers for stroke diagnosis [18, 19].

29

Kelly TJ, Souza AL, Clish CB, Puigserver P. A hypoxia -induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like.

In addition, miRNA-210 regulates survival via a positive feedback loop during hypoxia [16].

Recent studies have successfully established a functional link between cell survival and a discrete group of survival -regulating miRNAs, including miRNA-1 [14], miRNA-125 [15], miRNA-206 [14], miRNA-210 [16, 17] and miRNA-708 [18].

30

Nine miRNAs (miR-21, miR-24, miR-214, miR-132, miR-195, miR-210, miR-144, miR-150 and miR-34a) were found in exosomes obtained from rats subjected to RIPC, but only miR-24 was significantly upregulated ([#] P < 0.05, n = 4).

b, c Flow cytometric analysis of the uptake of exosomes by H9c2 cells at various time points We further determined the expression of nine miRNAs (miR-24, miR-21, miR-214, miR-132, miR-195, miR-210, miR-144, miR-150 and miR-34a) in both RIPC-EXO and EXO (Fig.   3a).

31

We suppose that SFI attenuates myocardial hypertrophy by upregulating the levels of particular miRNAs, including miR-19a-3p, miR-181d-5p, miR-210-3p, miR-352 and miR-324-3p, and downregulating miR-199a-5p.

32

According to the present study, several miRNAs were observed to target cholesterol metabolism -associated DEGs, including miR210, miR300-3p, miR-325-5p, miR-487b and miR-16.

In addition, a number of miRNAs, including miR-16, miR-210, miR-15b, miR300-3p, miR-540, miR-325-5p and miR-487b, were observed to have target DEGs involved in cholesterol -associated metabolism, e. g. IDI1 and FDFT1.

33

Up-regulation of miR-210 under hypoxic conditions confers cardioprotection in cultured cardiomyocytes [23, 24].

miR-210 and miR-150 protect the heart from ischemic injury by regulating cell death [16, 17].

34

Ischemia induces marked changes in microRNA expression and there is accumulating evidence that HIF-1α is responsible for regulating several miRNAs involved in cell responses to hypoxia, such as miR-210 or miR-373 [13].

35

In comparison, the elevated expression of miR-210 and miR-499 are more in favor with cell survival.

MiR-210 induces angiogenesis and miR-499 stimulates cardiac stem cells to commit into mature working cardiomyocytes, [49– 51] albeit cardiomyocytes are terminally differentiated in mature adult hearts and there are minimal cardiac stem cells to be stimulated.

36

For example, miR-21, miR-210 and miR-155 reduce pro-apoptotic signaling in response to a hypoxic environment and are consistently over-expressed in a variety of human tumors (Table 1).

We found that hypoxia related microRNA had the most significant fold changes, with miR-210, miR-155 and miR-21 being amongst the top.

37

Minayi et al. reported that miR-210 upregulates the proliferation of BM-MSCs [36].

38

Involvement of upregulation of miR-210 in a rat epilepsy mo del.

39

Previous studies showed that expression of four miRNAs, including miR-96, miR-193-3p, miR-210, and miR-21, were correlated with the skin flap mo del in rat [9].

In Figure 1G, the results of laser Doppler velocimetry showed that relatively low blood flow in Part C. Positive correlation between ischemia-reperfusion injury in the skin flap of rat and four miRNAs (miR-21, miR-96, miR-193, and miR-210) has been demonstrated in previous studies [9].

40

Among these differentially expressed miRNAs, rno-miR-130b-3p, rno-miR-132-3p, rno-miR-181a-1-3p, rno-miR-222-3p, rno-miR-351-5p, and rno-miR-21-3p had the largest positive fold changes, while rno-miR-335, rno-miR-192-3p, rno-miR-194-5p, rno-miR-192-5p, rno-miR-499-5p, and rno-miR-210-3p had the largest negative fold changes (Table 1).

41

miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b.

42

Only mouse Tcf7l2 has a validated miRNA for which it is the target—Mir210, which has only a human ortholog currently displayed in.

43

Some hypoxamirs, such as miR-210, have a similar expression pattern in ubiquitous types of cells 23, whereas others act as hypoxamirs only in specific types of tissues and cells.

44

Among these the miR-17–92 cluster, miR-21, miR-145, miR-204 and miR-210 are dysregulated in PASMCs, miR-124 is primarily dysregulated in fibroblasts in the adventitia, and miR-17, miR-21, miR-424, and miR-503 appear to play important roles in PAECs.

45

fcgi?artid=2785585&tool=pmcentrez&rendertype=abstract 6. Hu S, Huang M, Li Z, Jia F, Ghosh Z, Lijkwan M a, et al. MicroRNA-210 as a novel therapy for treatment of ischemic heart disease.

46

Chang W. Lee C. Y. Park J. H. Park M. S. Maeng L. S. Yoon C. S. Lee M. Y. Hwang K. C. Chung Y. A. Survival of hypoxic human mesenchymal stem cells is enhanced by a positive feedback loop involving mir-210 and hypoxia-inducible factor 1 J. Vet.

47

Johan et al. determined that circulating miR-210 levels significantly increased in critically ill patients with AKI and was an independent and powerful predictor of 28-day survival [15].

48

Visualization of exosome -mediated miR-210 transfer from hypoxic tumor cells.

49

miR-210: more than a silent player in hypoxia.

50

In addition, the neural progenitor cells isolated by LCM exhibited increases in miR-146a, miR-146b, miR-210, miR-19b and miR-378 and decreases in miR-128, miR-291a-3p, and miR-139-5p (Fig. 3A to 3C), which are consistent with the array data findings.

51

Lorenzen J. M. Kielstein J. T. Hafer C. Gupta S. K. Kumpers P. Faulhaber-Walter R. Haller H. Fliser D. Thum T. Circulating miR-210 predicts survival in critically ill patients with acute kidney injury Clin.

52

Co-administration of α-lipoic acid and cyclosporine aggravates colon ulceration of acetic acid -induced ulcerative colitis via facilitation of NO/COX-2/miR-210 cascade.

53

Other miRs which were more abundant in CDC-EVs vs MSC-EVs included miR-124, miR-210, miR-92 and miR-320.