目的:探讨miR-140-3p靶向调节PI3K/Akt通路对缺氧复氧心肌细胞再灌注损伤的影响。方法:对大鼠来源的H9C2心肌细胞进行缺氧/复氧以诱导细胞损伤,使用miR-140-3p mimics及其对照(NC)转染H9C2心肌细胞,再使用10 μmol/L LY294002处理过表达miR-140-3p的H9C2细胞。将心肌细胞分为6组,分别为对照组(CON组)、H/R组(Model组)、Model +NC组、Model +miR-140-3p mimics组、Model+miR-140-3p mimics + LY294002组、Model+ LY294002组。采用荧光定量PCR(RT-PCR)法检测各组miR-140-3p 相对表达量,CCK-8法检测各组心肌细胞活力,流式细胞仪检测各组心肌细胞凋亡率,Western blot检测各组磷酸化磷脂酰肌醇3激酶(p-PI3K)、磷酸化蛋白激酶B (p-Akt)、Bax、Bcl-2、Cleaved-caspases-3表达量。结果:与CON组比较,Model组miR-140-3p表达量、细胞活力以及Bcl-2、p-PI3K、p-Akt蛋白表达明显降低(P＜0.05),Bax、Cleaved-caspases-3蛋白表达和凋亡率明显提高(P＜0.05),与Model组比较,miR-140-3p过表达可上调miR-140-3p表达量、细胞活力以及Bcl-2、p-PI3K、p-Akt蛋白,下调Bax、Cleaved-caspases-3蛋白表达和凋亡率,LY294002可抑制miR-140-3p表达,下调细胞活力以及Bcl-2、p-PI3K、p-Akt蛋白,上调Bax、Cleaved-caspases-3蛋白表达和凋亡率。结论:MiR-140-3p可通过调节PI3K/Akt通路减轻缺氧复氧心肌细胞自噬和凋亡,进而减轻心肌损伤。
Objective: To investigate the effect of miR-140-3p on hypoxia/reoxygenation-induced myocardial cell reperfusion injury by targeting PI3K/Akt pathway. Methods: H9C2 cardiomyocytes derived from rats were subjected to hypoxia/reoxygenation to induce cell injury. MiR-140-3p mimics and their control (NC) were transfected into H9C2 cardiomyocytes, and H9C2 cells overexpressing miR-140-3p were treated with 10 μmol/L LY294002. The cardiomyocytes were divided into 6 groups, named control group (CON group), H/R group (Model group), Model+NC group, Model+miR-140-3p mimics group, Model+miR-140-3p mimics+LY294002 group, Model+LY294002 group. The relative expression of miR-140-3 p in each group was detected by fluorescence quantitative PCR (RT-PCR). The viability of cardiomyocytes in each group was detected by CCK-8 method. The apoptosis rate of cardiomyocytes in each group was detected by flow cytometry. The expression levels of phosphorylated phosphatidylinositol 3 kinase (p-PI3K), phosphorylated protein kinase B (p-Akt), Bax, Bcl-2, and cleared caspases-3 in each group were detected by Western blot. Results: Compared with the CON group, the expression level and cell viability of miR-140-3p, as well as the expression of Bcl-2, p-PI3K, and p-Akt proteins, were significantly reduced in the Model group (P＜0.05), while the expression and apoptosis rate of Bax and Cleared caspases-3 proteins were significantly increased (P＜0.05). Compared with the Model group, overexpression of miR-140-3p upregulated the expression level and cell viability of miR-140-3p, as well as Bcl-2, p-PI3K, and p-Akt proteins, while downregulated the expression and apoptosis rate of Bax and Cleared caspases-3 proteins, LY294002 could inhibit the expression of miR-140-3p, downregulate cell viability and Bcl-2, p-PI3K, p-Akt proteins, and upregulate the expression of Bax, Cleared caspases-3 proteins and apoptosis rate. Conclusion: MiR-140-3p can alleviate autophagy and apoptosis in hypoxia/reoxygenation cardiomyocytes by regulating the PI3K/Akt pathway, thereby alleviating myocardial injury.
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