While I/R is effective in restoring blood supply to ischemic tissue, it may also lead to additional damage, including the apoptosis and death of myocardial cells, resulting in cell loss and cardiac dysfunction . Thus, it is extremely important to devise methods of preventing and treating I/R injuries. It has been demonstrated that miRNAs are implicated in I/R damage[3, 13, 14].
Our findings are in agreement with those of several earlier cell and animal model studies that showed the protective roles of miRNA-19a and miRNA19b against the effects of hypoxia, ischemia, and heart failure resulting from endoplasmic stress; in all cases, the miRNAs reduced apoptosis. Here, in H9C2 cells, we found that H/R enhanced apoptosis and lower viability. Notably, H/R treatment reduced the miR-19a-3p levels. However, the effects of H/R on apoptosis were counteracted by elevated miRNA-19a-3p levels.
The miR-19a-3p overexpression was linked to a reduction in cell damage and apoptosis after H/R, suggesting the involvement of miR-19a-3p in the amelioration of myocardial I/R injuries. Overexpression of the miRNA also lowered the levels of proteins related to apoptosis. In contrast, increased damage, measured by reduced cell viability, together with increased apoptosis, measured by raised cleaved caspase 3 levels and elevated ratio of Bax/Bcl-2, was seen after inhibition of miR-19a-3p. This suggests that miR-19a-3p functions as an anti-apoptotic agent. Subsequent investigations indicated that miR-19a-3p targeted and decreased SOCS3 expression in H9C2 cells. Knockdown of SOCS3 also reduced H/R-induced apoptosis, while raised SOCS3 levels counteracted the mitigating action of miR-19a-3p on apoptosis. It thus appears that miR-19a-3p protects H/R-treated cells against apoptosis by modulation of SOCS3 signaling.
SOCS3 is the SOCS family member most closely associated with cardiovascular disease. It also modulates JAK-STAT signaling. Earlier studies have shown that myocardial STAT3 reduces apoptosis after I/R injury[16–18]. It is possible that SOCS3 knockout may activate STAT3 in the JAK-STAT3 pathway. STAT3 has been found to have a protective function in myocardial cells after AMI.
Hussain et al. reported that ghrelin has a cardioprotective effect in left ventricular injury induced by myocardial infarction, activating JAK2/STAT3 signaling through the inhibition of SOCS3. Our data suggest that H/R induces H9C2 cardiomyocyte injury by activating SOCS3/STAT3-mediated apoptosis. The miR-19a-3p downregulation seen after H/R also suggests the involvement of miR-19a-3p in H/R-induced cardiomyocyte injury.
To investigate this hypothesis, levels of miR-19a-3p were raised by transfection of a miR-19a-3p mimic. This significantly reduced apoptosis, associated with reduced levels of both SOCS3 and cleaved caspase 3. The abnormal expression of numerous miRNAs in myocardial I/R-injury models has been reported, indicating that miRNAs play significant roles in ischemic injury . miR-494 was shown to modulate PI3K/AKT/mTOR signaling by targeting SIRT1 and to protect cardiomyocytes from I/R injury by decreasing both apoptosis and autophagy. miRNA-15b downregulated both MAPK3 and Bcl-2 expression and aggravated cardiomyocyte apoptosis  while miR-181c-5p worsened H/R-induced cellular damage and apoptosis by regulating PTPN4, suggesting that targeting miR-181C-5p/PTPN4 signaling may reduce myocardial I/R injuries . In addition, miR-17-92 was found to mitigate kidney damage caused by H/R through several pathways . Our results suggest that miR-19a-3p protects against cardiac damage through the downregulation of SOCS3 and subsequent reduction in apoptosis. We, therefore, used various bioinformatics software packages, including miRanda, TargetScan, RNAhybrid, and target gene prediction at EMBL, for target prediction. Screening of the miR-19a-3p sequence indicated that it complemented the 3’ UTR of SOCS3. Luciferase assays in H9C2 cells confirmed that miR-19a-3p may interact with the 3'UTR of the human SOCS3 gene and thus inhibit its transcription. SOCS3 has been documented to regulate apoptosis in myocardial I/R injuries, and it was found that knockout of SOCS3 in cardiac tissue resulted in continuous activation of protective signaling pathways, leading to reductions in cell damage and apoptosis and suggesting the importance of SOCS3 in the aggravation of H/R-induced damage. In addition, reduced SOCS3 levels ameliorated the effects of ischemic preconditioning and mitigated myocardial H/R-induced damage . In summary, we observed that overexpression of SOCS3 reduced the protective miR-19a-3p effects on H/R-induced damage and apoptosis, while SOCS3 silencing enhanced cardiomyocyte protection and reduced apoptosis. As a result, our findings revealed that miR-19a-3p protects against damage induced by hypoxia by regulating SOCS3 to reduce apoptosis. Target prediction by bioinformatics software did not predict interactions between miR-19a-3p and cleaved caspase3, Bax, and Bcl-2.
In summary, this is the first demonstration that miR-19a-3p protects against hypoxia-induced cardiomyocyte injury through modulating the function of SOCS3. However, only the function of miR-19-3p was examined on apoptosis through SOCS3 and did not investigate the role of SOCS3-associated signaling in cardiomyocyte apoptosis. The study also only investigated cells in which H/R treatment was used to mimic I/R injuries. Further work is required to verify these results in animal models.