Miao Ying-Ying, Yuan Huan-Huan, Huang Min-Jie, Fu Sheng-Qi
Department of Anatomy, Sanquan College of Xinxiang Medical University, Xinxiang 453003, China.
Department of Human Anatomy and Histoembryology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang 453003, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2022 Jul;38(4):335-340. doi: 10.12047/j.cjap.6265.2022.064.
To investigate the effects of miRNA-130a-3p on autophagy and apoptosis induced by LPS in myocardial cells and its molecular mechanisms. H9C2 cells were divided into five groups: normal control group, LPS model group, miRNA negative control group miRNA-130a-3p mimics group(overexpression of miRNA-130a-3p) and miRNA-130a-3p mimics + LY294002 group(overexpression of miRNA-130a-3p + PI3K inhibitor). The LPS model group was induced by LPS at a final concentration of 10 μg/ml for 24 h. In the miRNA negative control group and miRNA-130a-3p mimics group, negative contro miRNA or miRNA-130a-3p mimics were transfected into H9C2 cells by lipo3000. After 24 h of culture, LPS was added into the medium for 24 hours. In the miRNA -130A-3P mimics + LY294002 group, miRNA -130A-3P mimics was transfected into H9C2 cells by using lipo3000, and LY294002 at a final concentration of 10 μmol/L was added to the culture medium for 24 h, followed by LPS at a concentration of 10 μg/ml for 24 h. The expression of miRNA-130a-3p mRNA in cells was detected by RT-qPCR. The CCK-8 assay was used to detect the cell viability. The contents of TNF-α, IL-6 and IL-1β were detected by ELISA assay. The contents of SOD and LDH in cell culture medium were detected by colorimetry. Western blot was used to detect the protein expressions of p-PI3K, p-AKT, Bax, Bcl-2, cleaved-caspase-3, LC3 and p62. The results showed that the levels of miRNA-130a-3p mRNA, p-PI3K protein and p-AKT protein in LPS model cells were significantly lower than those in normal control group(P<0.01), and the expressions of p-PI3K, p-AKT protein in miRNA-130a-3p mimics group were increased significantly compared with LPS group(P<0.01,P<0.05). Compared with normal control group, the cell viability was decreased significantly and the contents of TNF-α, IL-6, IL-1β and LDH were increased significantly(P<0.01), the contents of SOD was decreased significantly in LPS group(P<0.01). The protein expression levels of Bax, cleaved-caspase-3 and p62 were increased significantly, while the expression level of Bcl-2 and LC3II/I ratio were decreased significantly in LPS group(P<0.01). miRNA-130a-3p mimics could increase the cell viability, decrease the contents of TNF-α, IL-6, IL-1β and LDH(P<0.01,P<0.05), increase the contents of SOD(P<0.05), decrease the expressions of Bax, cleaved caspase-3, p62(P<0.01), promote the expression of Bcl-2(P<0.01) and increase the ratio of LC3II/I(P<0.05). Compared with miRNA-130a-3p mimics group, LY294002 reversed the effects of miRNA-130a-3p mimics on cells. Overexpression miRNA-130a-3p could partly promote autophagy and inhibit cell apoptosis by activating PI3K/AKT signaling pathway to alleviate LPS-induced myocardial injury.
探讨miRNA-130a-3p对脂多糖（LPS）诱导的心肌细胞自噬和凋亡的影响及其分子机制。将H9C2细胞分为五组：正常对照组、LPS模型组、miRNA阴性对照组、miRNA-130a-3p模拟物组（miRNA-130a-3p过表达）和miRNA-130a-3p模拟物+LY294002组（miRNA-130a-3p过表达+PI3K抑制剂）。LPS模型组用终浓度为10μg/ml的LPS诱导24小时。在miRNA阴性对照组和miRNA-130a-3p模拟物组中，通过脂质体3000将阴性对照miRNA或miRNA-130a-3p模拟物转染到H9C2细胞中。培养24小时后，向培养基中加入LPS 24小时。在miRNA -130A-3P模拟物+LY294002组中，用脂质体3000将miRNA -130A-3P模拟物转染到H9C2细胞中，并向培养基中加入终浓度为10μmol/L的LY294002 24小时，随后加入浓度为10μg/ml的LPS 24小时。通过RT-qPCR检测细胞中miRNA-130a-3p mRNA的表达。采用CCK-8法检测细胞活力。通过ELISA法检测TNF-α、IL-6和IL-1β的含量。用比色法检测细胞培养基中SOD和LDH的含量。采用蛋白质免疫印迹法检测p-PI3K、p-AKT、Bax、Bcl-2、裂解的caspase-3、LC3和p62蛋白的表达。结果显示，LPS模型细胞中miRNA-130a-3p mRNA、p-PI3K蛋白和p-AKT蛋白水平显著低于正常对照组（P<0.01），与LPS组相比，miRNA-130a-3p模拟物组中p-PI3K、p-AKT蛋白表达显著增加（P<0.01，P<0.05）。与正常对照组相比，LPS组细胞活力显著降低，TNF-α、IL-6、IL-1β和LDH含量显著增加（P<0.01），SOD含量显著降低（P<0.01）。LPS组中Bax、裂解的caspase-3和p62蛋白表达水平显著增加，而Bcl-2和LC3II/I比值显著降低（P<0.01）。miRNA-130a-3p模拟物可提高细胞活力，降低TNF-α、IL-6、IL-1β和LDH含量（P<0.01，P<0.05），增加SOD含量（P<0.05），降低Bax、裂解的caspase-3、p62表达（P<0.01），促进Bcl-2表达（P<0.01）并增加LC3II/I比值（P<0.05）。与miRNA-130a-3p模拟物组相比，LY294002逆转了miRNA-130a-3p模拟物对细胞的作用。过表达miRNA-130a-3p可通过激活PI3K/AKT信号通路部分促进自噬并抑制细胞凋亡，从而减轻LPS诱导的心肌损伤。
