Role of microRNA-130a in myocardial hypoxia/reoxygenation injury

Abstract

The aim of this study was to investigate the role of microRNA (miR)-130a in the pathogenesis of myocardial hypoxia/reoxygenation (H/R) injury. Primary rat cardiomyocytes were cultured and subjected to H/R treatment. Reverse transcription-quantitative polymerase chain reaction was performed to detect the levels of miR-130a, western blot analysis was used to determine the expression of various proteins, and CCK-8 assay was performed to determine cell viability. In addition, flow cytometry was used to assess apoptosis. The cell viability was significantly decreased and the apoptosis rate was significantly increased in H/R-treated primary cardiomyocytes, and the expression level of miR-130a was also elevated in these model cells. Transfection with miR-130a inhibitor significantly elevated the cell viability and reduced the apoptosis rate in H/R-treated cardiomyocytes. Bioinformatics analysis indicated that autophagy-related gene 14 (ATG14) is the target for miR-130a, which was confirmed by dual-luciferase reporter assay and western blot analysis. When the H/R model cells were co-transfected with miR-130a inhibitor and small interfering RNA against ATG14, the cell viability was significantly reduced and the apoptosis rate was significantly elevated, compared with that of cells transfected with miR-130a inhibitor alone. miR-130a inhibitor transfection significantly elevated the levels of ATG14 and phosphorylated (p-)Beclin 1, increased the LC3II/LC3I ratio, and decreased the expression levels of P62 and cleaved caspase-3, while the co-transfection of miR-130a inhibitor and siR-ATG14 attenuated these effects in H/R-induced primary cardiomyocytes. These results indicate that miR-130a is involved in H/R-induced injuries in primary cardiomyocytes, and that the inhibition of miR-130a increases the levels of ATG14 and p-Beclin 1, thereby increasing autophagy and inhibiting apoptosis in these cells.

Keywords: apoptosis; autophagy; autophagy-related gene 14; hypoxia/reoxygenation; miR–130a; microRNA.

Figure 1.

Involvement of miR–130a in H/R-induced injuries in primary cardiomyocytes. (A) Cell viability and (B) apoptosis of H/R-induced cardiomyocytes transfected with 100 nM miR–130a inhibitor were assessed. (C) The expression level of miR–130a in H/R-induced cardiomyocytes was detected using RT-qPCR. (D) Primary cardiomyocytes were transfected with miR–130a inhibitor at 25, 50, and 100 nM, respectively, and RT-qPCR was performed to detect the expression levels of miR–130a in these cells. (E) Representative results for the flow cytometry to detect apoptosis. **P<0.01 vs. the normal control group; ^#P<0.05 vs. H/R-induced model cells. miR, microRNA; H/R, hypoxia/reoxygenation; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control; inh, inhibitor.

Figure 2.

Interaction between miR–130a and ATG14. (A) Prediction of miR–130a target gene with TargetScan software. (B) Dual-luciferase reporter assay results showing the interaction between miR–130a and ATG14. (C) The expression levels of ATG14 as detected by western blot analysis. *P<0.01 vs. the NC group. miR, microRNA; ATG14, autophagy-related gene 14; UTR, untranslated region; wt, wild type; mut, mutant; NC, negative control.

Figure 3.

Effects of ATG14 interference on cell viability and apoptosis in H/R-induced primary cardiomyocytes. (A) Primary cardiomyocytes were transfected with siR-ATG14 at 25, 50, and 100 nM, respectively, and the mRNA expression levels of ATG14 were detected with RT-qPCR. The H/R-induced primary cardiomyocytes were co-transfected with miR–130a inhibitor and siR-ATG14, and then (B) the cell viability and (C) apoptosis were assessed using CCK-8 assay and flow cytometry, respectively. (D) Representative results for the flow cytometric detection of apoptosis. **P<0.01 vs. the NC group; ^#P<0.05 vs. the H/R-induced model cells; ^&P<0.05 vs. the H/R models treated with miR–130a inhibitor. ATG14, autophagy-related gene 14; H/R, hypoxia/reoxygenation; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siR, small interfering RNA; NC, negative control; inh, inhibitor.

Figure 4.

Effects of miR–130a inhibition on autophagy and apoptosis in H/R-induced primary cardiomyocytes. Western blot analysis was performed to detect the expression levels of ATG14, p-Beclin 1, LC3, P62 and c-CASP3 in the H/R-induced primary cardiomyocytes transfected with miR–130a inhibitor and/or siR-ATG14. miR, microRNA; H/R, hypoxia/reoxygenation; ATG14, autophagy-related gene 14; p-Beclin 1, phosphorylated Beclin 1; c-CASP3, cleaved caspase-3; inh, inhibitor.