Department of Anesthesiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China.
Int J Mol Med. 2019 Jan;43(1):143-154. doi: 10.3892/ijmm.2018.3988. Epub 2018 Nov 12.
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 cardiomyocytes 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 downregulation 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.
本研究旨在探讨微小 RNA-181b-5p（miR-181b-5p）通过靶向热休克蛋白家族 A 成员 5（Hspa5）在饥饿诱导的心肌细胞自噬中的作用。为此，将 H9c2 心肌细胞和乳鼠心室肌细胞（NRVMs）在 Earle 的平衡盐溶液（EBSS）中饥饿不同时间（0、2、4、6 和 8 h）。通过 RT-qPCR 分析检测各组中 miR-181b-5p 的表达。通过免疫荧光法检测 LC3 的表达。此外，将 miR-181b-5p 模拟物、miR-181b-5p 抑制剂、siHspa5 或其各自的对照转染至 H9c2 心肌细胞和 NRVMs 中。通过 MTT 测定法检测各组细胞增殖情况。通过 Western blot 分析检测 Beclin-1、Hspa5、磷酸化磷脂酰肌醇 3-激酶 PI3K（p-PI3K）、磷酸化 Akt（p-Akt）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、Bcl-2、Bax 和 cleaved caspase-3 的表达。通过流式细胞术评估细胞凋亡。通过荧光素酶报告基因测定法确定 Hspa5 是否为 miR-181b-5p 的直接靶标。结果显示，miR-181b-5p 的下调促进了心肌细胞中的细胞自噬。此外，miR-181b-5p 负调控 Beclin-1 和 Hspa5。Beclin-1 是一种众所周知的与自噬和细胞凋亡相关的蛋白。此外，miR-181b-5p 在心肌细胞中的表达下调可减轻细胞凋亡。Bcl-2 通过分别与 Bax 和 Bcl-2 结合来防止凋亡和自噬。miR-181b-5p 的上调通过 Hspa5 抑制自噬并促进凋亡。miR-181b-5p 抑制通过 Hspa5 抑制自噬并促进凋亡可促进 p-mTOR、p-Akt 和 p-PI3K 的表达。荧光素酶报告基因测定的结果也证实 Hspa5 是 miR-181b-5p 的直接靶标。总的来说，本研究结果表明，miR-181b-5p 通过 PI3K/Akt/mTOR 信号通路直接靶向 Hspa5 促进饥饿诱导的心肌细胞自噬和凋亡。
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