MicroRNA-489 promotes cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion injury through inhibiting SPIN1

Y. Chen, S. Wu, X.-S. Zhang, D.-M. Wang, C.-Y. Qian

Department of Emergency, The First People’s Hospital of Changzhou, The Third Affiliated Hospital of Soochow University, Changzhou, China

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• Cardiology

OBJECTIVE: To investigate whether microRNA-489 could promote cardiomyocyte apoptosis through targeting inhibition of SPIN1, thus participating in the development of myocardial ischemia-reperfusion injury.
MATERIALS AND METHODS: MicroRNA-489 expression in H9c2 cells induced with hypoxia/reoxygenation (H/R) was determined by quantitative real-time polymerase chain reaction (qRT-PCR). With microRNA-489 overexpression in H/R H9c2 cells, activities of lactate dehydrogenase (LDH), methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were detected using relative commercial kits, respectively. The regulatory effect of microRNA-489 on the proliferation and apoptosis of H/R H9c2 cells was assessed through cell counting kit-8 (CCK-8) assay and flow cytometry (FCM), respectively. Through conducting a dual-luciferase reporter gene assay, we evaluated the binding condition between microRNA-489 and SPIN1. Protein expressions of apoptotic genes in H/R H9c2 cells with microRNA-489 overexpression were determined by Western blot. Finally, the regulatory role of microRNA-489 in PI3K/AKT pathway was detected through Western blot.
RESULTS: QRT-PCR data showed that microRNA-489 was highly expressed in H/R H9c2 cells than those in normoxic control. Overexpression of microRNA-489 in H/R H9c2 cells increased the activities of LDH, MDA and GSH-PX, while decreased the activities of SOD. MicroRNA-489 overexpression markedly inhibited the proliferative rate but accelerated apoptosis of H/R H9c2 cells. Western blot results indicated that protein expressions of pro-apoptotic genes Bax and cytochrome C upregulated, whereas anti-apoptotic gene Bcl-2 downregulated after overexpression of microRNA-489 in H/R H9c2 cells. We confirmed that microRNA-489 could target to SPIN1 and inhibit its expression. After overexpression of microRNA-489 in H/R H9c2 cells, PI3K/AKT pathway was inhibited, which was further reversed by PI3K/AKT pathway agonist SC79. Besides, SC79 treatment also reversed the regulatory effect of overexpressed microRNA-489 on cellular behaviors of H/R H9c2 cells.
CONCLUSIONS: MicroRNA-489 inhibits the proliferation and accelerates apoptosis of cardiomyocytes after MIRI by targeting inhibition of SPIN1 via inactivating PI3K/AKT pathway. MicroRNA-489 may be a potential therapeutic target for MIRI.

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