The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Int J Med Sci. 2020 Sep 16;17(16):2570-2577. doi: 10.7150/ijms.45408. eCollection 2020.
X-inactive specific transcript (Xist) is a lncRNA, which plays a significant role in X-chromosome inactivation, regulates cell proliferation in tumor cells, and inhibits apoptosis in acute myocardial infarction. On the other hand, miR-7a-5p is involved in cardiomyocytes injury in myocardial ischemia/reperfusion. However, their roles in LPS-induced damage remain unclear. This study aimed at using siRNA transfection and lentivirus infection to regulate the expression of xist and miR-7a-5p, and to evaluate their effects on LPS-induced myocardial damage. Mice cardiomyocytes (MCM) cells were divided into six groups, namely the control group, the LPS group, the LPS + lncRNA group, the LPS + lncRNA group, the LPS + miRNA group, and the LPS + miRNA group. Quantitative real-time PCR (qRT-PCR) was performed to assay for the RNA expressions of xist, miR-7a-5p, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and recombinant mitochondrial transcription factor A (Tfam) in all the groups. The ATP level was determined using the adenosine triphosphate (ATP) assay kit according to the manufacturer's instructions. Flow cytometry was performed to estimate the level of apoptosis and proliferation in cells in each group. The level of xist in the myocardial cells was markedly higher in the LPS group compared with the control group; however, it was reduced in the LPS+ lncRNA group. There was no significant difference in the expression of xist among the LPS+miRNA, LPS+miRNA, and LPS groups. Moreover, the expression of mir-7a-5p was significantly reduced in myocardial cells in the LPS group, and moderately reduced in the LPS+ miRNA group, but remarkably elevated in the LPS+ miRNA group (P<0.05). The expression of mir-7a-5p was comparably similar in the LPS+ lncRNA group, LPS+ lncRNA group, and LPS groups. Further, the levels of PGC-1a, and Tfam were determined. In the LPS group, the expression of PGC-1α was significantly reduced but elevated in the LPS+lncRNA and LPS+ miRNA groups (P<0.05). There was no significant difference in the level of PGC-1α among the LPS, LPS+ lncRNA, and LPS+ miRNA groups. The expression of Tfam was markedly reduced in the LPS group ( 0.05), but elevated after the suppression of xist and mir-7a-5p. The expression of Tfam was not significantly different among the LPS group, LPS+ lncRNA and LPS+ miRNA groups. Notably, overexpression of mir-7a-5p had a mild effect on the expression of Tfam in the LPS+ miRNA group compared with the control group. Besides, ATP expression in the LPS group was markedly reduced, but elevated after the inhibition of xist and mir-7a-5p. Suppressing the expression of xist or mir-7a-5p resulted in reduced cell apoptosis and increased cell proliferation. In this study, we established that down-regulation of xist and mir-7a-5p reduces apoptosis in response to LPS.
X 失活特异性转录物（Xist）是一种长链非编码 RNA，在 X 染色体失活、调节肿瘤细胞增殖和抑制急性心肌梗死后细胞凋亡中发挥重要作用。另一方面，miR-7a-5p 参与心肌缺血/再灌注中的心肌细胞损伤。然而，它们在 LPS 诱导的损伤中的作用尚不清楚。本研究旨在通过 siRNA 转染和慢病毒感染来调节 xist 和 miR-7a-5p 的表达，并评估它们对 LPS 诱导的心肌损伤的影响。将小鼠心肌细胞（MCM）细胞分为六组，即对照组、LPS 组、LPS+lncRNA 组、LPS+lncRNA 组、LPS+miRNA 组和 LPS+miRNA 组。采用定量实时 PCR（qRT-PCR）检测各组细胞中 xist、miR-7a-5p、过氧化物酶体增殖物激活受体-γ共激活因子-1α（PGC-1α）和重组线粒体转录因子 A（Tfam）的 RNA 表达水平。根据制造商的说明，使用三磷酸腺苷（ATP）测定试剂盒测定 ATP 水平。通过流式细胞术评估各组细胞的凋亡和增殖水平。LPS 组心肌细胞中 xist 的水平明显高于对照组；然而，在 LPS+lncRNA 组中，xist 的表达减少。LPS+miRNA、LPS+miRNA 和 LPS 组之间 xist 的表达无显著差异。此外，miR-7a-5p 在 LPS 组心肌细胞中的表达显著降低，在 LPS+miRNA 组中适度降低，但在 LPS+miRNA 组中显著升高（P<0.05）。LPS+lncRNA 组、LPS+lncRNA 组和 LPS 组之间 miR-7a-5p 的表达相似。进一步测定了 PGC-1α 和 Tfam 的水平。在 LPS 组中，PGC-1α 的表达显著降低，但在 LPS+lncRNA 和 LPS+miRNA 组中升高（P<0.05）。LPS、LPS+lncRNA 和 LPS+miRNA 组之间 PGC-1α 的水平无显著差异。Tfam 的表达在 LPS 组明显降低（P<0.05），但在抑制 xist 和 mir-7a-5p 后升高。LPS 组、LPS+lncRNA 组和 LPS+miRNA 组之间 Tfam 的表达无显著差异。值得注意的是，与对照组相比，miR-7a-5p 的过表达对 LPS+miRNA 组中 Tfam 的表达仅有轻度影响。此外，LPS 组的 ATP 表达明显降低，但抑制 xist 和 mir-7a-5p 后升高。抑制 xist 或 mir-7a-5p 的表达导致细胞凋亡减少和细胞增殖增加。本研究表明，下调 xist 和 mir-7a-5p 可减少 LPS 诱导的细胞凋亡。
