Abstract
BackgroundOxidative stress results in cell apoptosis/death and plays a detrimental role in disease development and progression. Stressors alter the miRNA expression profile and miRNAs play a role in the cell response to stress. We previously showed that miR-711 is significantly over-expressed in extended cold ischemia reperfusion injured hearts in heart transplant. In this study, we aimed to investigate the role of miR-711 in cardiac cell damage in response to oxidative stress and how miR-711 is regulated.MethodsRat cardiac cell line H9c2 cells were cultured and exposed to oxidative conditions (Antimycin A (AA), H2O2, CoCl2, or cold hypoxia/reoxygenation (H/R)) in vitro. H9c2 cells were transfected with miR-711 mimics, miR-711 inhibitors, or small interference RNA, using transfection reagents. The expression of miR-711 was measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cell apoptosis/death was detected by flow cytometry and an IncuCyte system. Mitochondrial damage was detected by measuring the mitochondria membrane potential by flow cytometry. Gene expression was detected by qRT-PCR at the mRNA level and Western blotting and immunocytochemistry staining at the protein level.ResultsWe found that miR-711 was significantly up-regulated in cells treated with H2O2, AA, CoCl2, and cold H/R. Over-expression of miR-711 increased cell apoptosis/death induced by AA and H/R whereas cell death was reduced by miR-711 inhibitors. MiR-711 induced cell death through negative regulation of angiopoietin 1 (Ang-1), fibroblast growth factor 14 (FGF14) and calcium voltage-gated channel subunit alpha1C (Cacna1c) genes. Both knockdown of hypoxia inducible factor 1α (HIF-1α) and inactivation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFКB) pathway inhibited over-expression of miR-711.ConclusionOxidative stress increases the expression of miR-711. Over-expression of miR-711 induces cell apoptosis/death. HIF-1α and NFКB regulate miR-711 in H9c2 cells during oxidative stress. miR-711 is a new target for preventing oxidative stress.
Highlights
Oxidative stress results in cell apoptosis/death and plays a detrimental role in disease development and progression
We found that microRNA 711 (miR-711) in cells treated with H2O2 at all tested concentrations was upregulated compared with the untreated control (Fig. 1a left), despite the difference of miR-711 expression between 150 μM H2O2 and control was not statistically significant
The results showed that treatment with 300 μM H2O2 for 6 h did not significantly increase the expression of miR-711, whereas both treatment for 12 h and 24 h significantly up-regulated miR-711 (Fig. 1a right)
Summary
Oxidative stress results in cell apoptosis/death and plays a detrimental role in disease development and progression. We previously demonstrated that the expression of miRNAs is dysregulated in I/R injured heart cells and that miR-711 is significantly up-regulated with the greatest fold change [21]. Recent studies have shown that miR-711 is up-regulated in trauma injured brains and ischemic heart cells and that its over-expression causes cell death [22,23,24,25]. MiR-711 is involved in HIV infection [26] and cancer [27, 28] It is currently unknown how miR-711 is regulated during oxidative stress and by what molecular mechanism miR-711 causes cell death in response to oxidative stress
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