miR-633通过调节蛋白激酶B影响缺氧诱导H9C2细胞生物学功能研究

The miR-633 affects the biological function of hypoxia-induced H9C2 cells by regulating protein kinase B

目的探讨miR-633对缺氧诱导的H9C2细胞生物学功能调节作用。方法缺氧诱导培养H9C2细胞,利用实时定量荧光PCR检测缺氧后细胞中miR-633表达情况。通过MTT实验和Transwell实验检测缺氧对细胞增殖和迁移的调节作用。在缺氧H9C2细胞中下调miR-633的表达,通过MTT实验和Transwell实验检测miR-633对缺氧后细胞增殖和迁移的调节作用。miRDB软件预测miR-633可能打靶的蛋白,荧光素酶报告基因实验分析miR-633对蛋白激酶B(AKT)的靶向调节作用。通过Western blot检测miR-633对缺氧后H9C2细胞中AKT、细胞周期素D1(Cyclin D1)、B淋巴细胞瘤-2(bcl-2)与基质金属蛋白酶2(MMP2)的影响。结果缺氧诱导后H9C2细胞中miR-633表达上调,缺氧诱导抑制H9C2细胞的增殖和迁移能力。MTT和Transwell实验证明,miR-633受到抑制后能够显著促进H9C2细胞的增殖和迁移。miR-633与AKT的3′UTR区域具有结合位点,miR-633可以抑制AKT的生物学活性,下调miR-633能够促进AKT、Cyclin D1、bcl-2与MMP2蛋白的表达。结论 miR-633可以通过调节AKT的活性促进缺氧诱导的H9C2细胞的增殖与迁移。

Objective To investigate the regulation of miR-633 on the biological function of hypoxia-induced H9 C2 cells.Methods H9 C2 cells were cultured under hypoxia induction,and the expression of miR-633 in hypoxia cells was detected by Real-time PCR.The effects of hypoxia on cell proliferation and migration were examined by MTT assay and Transwell assay.The expression of miR-633 was down-regulated in hypoxic H9 C2 cells,and the regulatory effect of miR-633 on the proliferation and migration of hypoxic cells was detected by MTT assay and Transwell assay.The miRDB software predicted the potential target proteins of miR-633,and luciferase reporter gene assay analyzed the targeting regulatory effect of miR-633 on protein kinase B(AKT).The effects of miR-633 on AKT,Cyclin D1,b-lymphoblastoma-2(bcl-2)and matrix metalloproteinase 2(MMP2)in hypoxic H9 C2 cells were determined by Western blot.Results After hypoxia induction,miR-633 expression in H9 C2 cells was up-regulated,and hypoxia induction inhibited the proliferation and migration ability of H9 C2 cells.MTT and transwell experiments showed that miR-633 inhibition significantly promoted the proliferation and migration of H9 C2 cells.The miR-633 has binding sites in the 3’UTR region of AKT.The miR-633 can inhibit the biological activity of AKT,and down-regulation of miR-633 can promote the expression of AKT,Cyclin D1,bcl-2 and MMP2.Conclusion The miR-633 can promote hypoxia-induced proliferation and migration of H9 C2 cells by regulating AKT activity.