MiR-181b-5p suppresses starvation-induced cardiomyocyte autophagy by targeting Hspa5.

Abstract

This study aimed to investigate the role of microRNA-181b-5p (miR-181b-5p) in starvation-induced cardiomyocyte autophagy by targeting heat shock protein family A member 5 (Hspa5). For this purpose, H9c2 cardiomyocytes and neonatal rat ventricular myocytes (NRVMs) were glucose-starved in Earle's Balanced Salt Solution (EBSS) for different periods of time (0, 2, 4, 6 and 8 h). RT-qPCR analysis was performed to examine the expression of miR-181b-5p in the different groups. Immunofluorescence was performed to detect the expression of LC3. In addition, the H9c2 cardiomyo-cytes and NRVMs were transfected with miR-181b-5p mimic, miR-181b-5p inhibitor, siHspa5 or their respective controls. An MTT assay was performed to measure cell proliferation in the different groups. Western blot analysis was performed to determine the expression of Beclin-1, Hspa5, phosphorylated phosphoinositide 3-kinase PI3K (p-PI3K), phosphorylated Akt (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), Bcl-2, Bax and cleaved caspase-3. Flow cytometry was performed to assess cell apoptosis. A luciferase reporter assay was performed to determine whether Hspa5 is a direct target of miR-181b-5p. The results revealed that the down-regulation of miR-181b-5p promoted cell autophagy in the cardiomyocytes. Moreover, miR-181b-5p negatively regulated Beclin-1 and Hspa5. Beclin-1 is a well-known autophagy- and apoptosis-related protein. In addition, cell apoptosis was attenuated by the decreased expression of miR-181b-5p in the cardiomyocytes. Bcl-2 prevented apoptosis and autophagy by binding to Bax and Bcl-2, respectively. The upregulation of miR-181b-5p inhibited autophagy and promoted apoptosis via Hspa5. miR-181b-5p inhibition promoted p-mTOR, p-Akt and p-PI3K expression via Hspa5. The results of luciferase reporter assay also confirmed that Hspa5 is a direct target of miR-181b-5p. On the whole, the findings of this study suggest that miR-181b-5p contributes to starvation-induced autophagy and apoptosis in cardiomyocytes by directly targeting Hspa5 via the PI3K/Akt/mTOR signaling pathway.