Liu Hongyan, Huan Lei, Yin Jie, Qin Meiling, Zhang Zengtang, Zhang Zhiqiang, Zhang Junye, Wang Shu
Department of Cardiology, Laiwu City People's Hospital, Laiwu, Shandong 271100, P.R. China.
Department of Endocrinology, Traditional Chinese Medical Hospital of Laiwu, Laiwu, Shandong 271100, P.R. China.
Exp Ther Med. 2017 Feb;13(2):759-765. doi: 10.3892/etm.2016.3984. Epub 2016 Dec 19.
The aim of this study was to investigate the role of microRNA (miR)-130a in the pathogenesis of myocardial hypoxia/reoxygenation (H/R) injury. Primary rat cardiomyocytes were cultured and subjected to H/R treatment. Reverse transcription-quantitative polymerase chain reaction was performed to detect the levels of miR-130a, western blot analysis was used to determine the expression of various proteins, and CCK-8 assay was performed to determine cell viability. In addition, flow cytometry was used to assess apoptosis. The cell viability was significantly decreased and the apoptosis rate was significantly increased in H/R-treated primary cardiomyocytes, and the expression level of miR-130a was also elevated in these model cells. Transfection with miR-130a inhibitor significantly elevated the cell viability and reduced the apoptosis rate in H/R-treated cardiomyocytes. Bioinformatics analysis indicated that autophagy-related gene 14 (ATG14) is the target for miR-130a, which was confirmed by dual-luciferase reporter assay and western blot analysis. When the H/R model cells were co-transfected with miR-130a inhibitor and small interfering RNA against ATG14, the cell viability was significantly reduced and the apoptosis rate was significantly elevated, compared with that of cells transfected with miR-130a inhibitor alone. miR-130a inhibitor transfection significantly elevated the levels of ATG14 and phosphorylated (p-)Beclin 1, increased the LC3II/LC3I ratio, and decreased the expression levels of P62 and cleaved caspase-3, while the co-transfection of miR-130a inhibitor and siR-ATG14 attenuated these effects in H/R-induced primary cardiomyocytes. These results indicate that miR-130a is involved in H/R-induced injuries in primary cardiomyocytes, and that the inhibition of miR-130a increases the levels of ATG14 and p-Beclin 1, thereby increasing autophagy and inhibiting apoptosis in these cells.
本研究旨在探讨微小RNA(miR)-130a在心肌缺氧/复氧(H/R)损伤发病机制中的作用。培养原代大鼠心肌细胞并进行H/R处理。采用逆转录-定量聚合酶链反应检测miR-130a水平,蛋白质印迹分析用于测定各种蛋白质的表达,CCK-8法用于测定细胞活力。此外,采用流式细胞术评估细胞凋亡。H/R处理的原代心肌细胞的细胞活力显著降低,凋亡率显著升高,且这些模型细胞中miR-130a的表达水平也升高。用miR-130a抑制剂转染可显著提高H/R处理的心肌细胞的细胞活力并降低凋亡率。生物信息学分析表明,自噬相关基因14(ATG14)是miR-130a的靶标,双荧光素酶报告基因检测和蛋白质印迹分析证实了这一点。当H/R模型细胞与miR-130a抑制剂和针对ATG14的小干扰RNA共转染时,与单独用miR-130a抑制剂转染的细胞相比,细胞活力显著降低,凋亡率显著升高。miR-130a抑制剂转染显著提高了ATG14和磷酸化(p-)Beclin 1的水平,增加了LC3II/LC3I比值,并降低了P62和裂解的caspase-3的表达水平,而miR-130a抑制剂和siR-ATG14的共转染减弱了H/R诱导的原代心肌细胞中的这些作用。这些结果表明,miR-130a参与了原代心肌细胞的H/R诱导损伤,抑制miR-130a可提高ATG14和p-Beclin 1的水平,从而增加这些细胞的自噬并抑制凋亡。