miR-1通过PI3K/Akt/mTOR信号通路对高糖诱导的H9c2心肌细胞自噬的影响

目的 探讨miR-1对高糖诱导的H9c2心肌细胞自噬及磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路的影响。方法H9c2细胞随机分为4组。正常组使用普通培养基培养，未转染；诱导组使用含33 mmol/L葡萄糖的培养基诱导，未转染;miR-1 NC组使用含33 mmol/L葡萄糖的培养基诱导，转染miR-1 NC；miR-1 inhibitor组使用含33 mmol/L葡萄糖的培养基诱导，转染miR-1 inhibitor。在加入葡萄糖诱导前48 h，使用Lipofectamine 3000转染试剂盒进行转染。实时荧光定量聚合酶链式反应（qRT-PCR）检测H9c2细胞和高糖诱导的H9c2细胞中miR-1表达水平；CCK-8法检测各组H9c2细胞增殖活性；蛋白免疫印记法（Western blot）检测各组H9c2细胞中Beclin1、p65、PI3K蛋白表达水平、LC3-Ⅱ/LC3-Ⅰ比值以及AKT、mTOR蛋白磷酸化水平；生物信息学预测和双萤光素酶验证miR-1和PI3K的靶向关系。结果与正常组相比，诱导组的H9c2细胞中，miR-1表达水平显著升高（P<0.05），PI3K蛋白表达水平显著降低（P<0.05）。与正常组相比，诱导组H9c2细胞Beclin1蛋白表达、LC3-Ⅱ/LC3-Ⅰ比值显著升高，自噬小体增多，细胞增殖率、p62蛋白表达以及PI3K、Akt、mTOR蛋白磷酸化水平显著降低（P<0.05）；与诱导组和miR-1 NC组相比，miR-1 inhibitor组H9c2细胞miR-1水平、Beclin1蛋白表达、LC3-Ⅱ/LC3-Ⅰ比值显著降低，自噬小体减少，细胞增殖率、p62蛋白表达以及PI3K、Akt、mTOR蛋白磷酸化水平显著升高（P<0.05）；miR-1和PI3K间存在靶向关系。结论在高糖诱导的H9c2细胞中，miR-1表达水平升高，抑制miR-1，可通过靶向激活PI3K/Akt/mTOR信号通路，抑制高糖诱导的H9c2心肌细胞自噬。

Effects of miR-1 on high glucose-induced autophagy in H9c2 cardiomyocytes via PI3K/Akt/mTOR signaling pathway

Objective To investigate the effect of miR-1 on high glucose-induced autophagy in H9c2 cardiomyocytes and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Methods H9c2 cells were randomly divided into 4 groups, normal group cultured in normal medium without transfection; induction group induced with 33 mmol/L glucose without transfection; miR-1 NC group induced with 33 mmol/L glucose and transfected with miR-1 NC; and miR-1 inhibitor group induced with 33 mmol/L glucose and transfected with miR-1 inhibitor. Forty-eight hours before glucose induction, Lipofectamine 3000 transfection kit was used for transfection. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of miR-1 in H9c2 cells and high glucose-induced H9c2 cells. CCK-8 method was used to detect the proliferation activity of H9c2 cells. Western blot was used to detect the protein expression levels of Beclin1, p65 and PI3K, LC3-Ⅱ/LC3-Ⅰratio, protein phosphorylation levels of Akt and mTOR in H9c2 cells.Bioinformatics prediction and double luciferase were used to verify the targeting relationship between miR-1 and PI3K. Results Compared with normal group, the miR-1 expression level in H9c2 cells of induction group was significantly higher (P<0.05), and the PI3K protein expression level was significantly lower (P<0.05). Compared with those in normal group, in the induction group Beclin1 protein expression and LC3-Ⅱ/LC3-Ⅰratio of H9c2 cells were significantly higher; autophagosomes was significantly increased; cell proliferation rate, p62 protein expression, and PI3K, Akt and mTOR protein phosphorylation levels were significantly lower (P<0.05).Compared with those in induction group and miR-1 NC group, the miR-1 level, in miR-1 inhibitor group Beclin1 protein expression and LC3-Ⅱ/LC3-Ⅰratio of H9c2 cells were significantly lower; autophagosomes was significantly decreased; cell proliferation rate, p62 protein expression, and PI3K, Akt and mTOR protein phosphorylation levels were significantly higher (P<0.05).There was a targeting relationship between miR-1 and PI3K. Conclusion In H9c2 cells induced by high glucose, the expression level of miR-1 is increased and the inhibition of miR-1 can inhibit the autophagy of H9c2 cardiomyocytes induced by high glucose via activating PI3K/Akt/mTOR signaling pathway.