miR - 93 - 5p抑制呼吸道合胞病毒感染支气管上皮细胞凋亡和炎症反应的机制研究

目的 研究miR - 93 - 5p是否通过靶向Mg2+/Mn2+依赖性蛋白磷酸酶1A（PPM1A）基因抑制呼吸道合胞病毒（RSV）感染的支气管上皮细胞凋亡和炎症反应。方法 将人支气管上皮细胞16 - HBE分为NC组、RSV组、miR - NC+RSV组、miR - 93 - 5p + RSV组、si - NC + RSV组、si - PPM1A + RSV组、pcDNA - NC + RSV组、pcDNA - PPM1A + RSV组、miR - 93 - 5p + pcDNA - NC + RSV组、miR - 93 - 5p + pcDNA - PPM1A + RSV组，利用脂质体Lipofectamine 2000进行miR - NC、miR - 93 - 5p、si - NC、si - PPM1A、pcDNA - NC、pcDNA - PPM1A的转染，并以RSV感染细胞。应用实时荧光定量PCR检测miR - 93 - 5p和PPM1A mRNA表达，western blot测定PPM1A、Cleaved - caspase - 3蛋白表达，ELISA分析TNF - α、IL - 6和IL - 1α分泌，流式细胞仪评估细胞凋亡。生物信息学预测与荧光素酶活性检测验证miR - 93 - 5p对PPM1A的靶向调控。结果 与NC组比较，RSV组细胞16 - HBE中miR - 93 - 5p表达量减少，PPM1A mRNA和PPM1A蛋白、Cleaved - caspase - 3蛋白表达量、TNF - α、IL - 6、IL - 1α分泌和细胞凋亡率增加。miR - 93 - 5p + RSV组细胞16 - HBE中Cleaved - caspase - 3蛋白水平、TNF - α、IL - 6、IL - 1α分泌和细胞凋亡率低于miR - NC + RSV组。miR - 93 - 5p靶向调控PPM1A的表达。与si - NC + RSV组比较，si - PPM1A + RSV组细胞16 - HBE中Cleaved - caspase - 3蛋白水平、TNF - α、IL - 6、IL - 1α分泌和细胞凋亡率降低，而pcDNA - PPM1A + RSV组结果与之相反。与miR - 93 - 5p + pcDNA - NC + RSV组比较，miR - 93 - 5p + pcDNA - PPM1A + RSV组提高细胞16 - HBE的Cleaved - caspase - 3蛋白水平、TNF - α、IL - 6、IL - 1α分泌、细胞凋亡率。上述差异均有统计学意义（均P＜0.05）。结论 miR - 93 - 5p通过靶向PPM1A基因，抑制RSV感染的支气管上皮细胞凋亡和炎症反应。

Mechanism of miR-93-5p inhibiting respiratory syncytial virus-infected bronchial epithelial cell apoptosis and inflammation

Abstract: Objective To investigate whether miR-93-5p can inhibit the apoptosis and inflammation of bronchial epithelial cells infected by respiratory syncytial virus (RSV) by targeting Mg2+/Mn2+-dependent protein phosphatase 1A (PPM1A) gene. Methods Human bronchial epithelial cells 16-HBE were divided into NC group, RSV group, miR-NC+RSV group, miR-93-5p+RSV group, si-NC+RSV group, si-PPM1A+RSV group, pcDNA-NC+RSV group, pcDNA-PPM1A+RSV group, miR-93-5p+pcDNA-NC+RSV group, miR-93-5p+pcDNA-PPM1A+RSV group, using Lipofectamine 2000 for miR-NC, miR-93-5p,miR-93-5p, si-PPM1A, pcDNA-NC, pcDNA-PPM1A transfection, and infection of cells with RSV. Real-time quantitative PCR was used to detect the expression of miR-93-5p and PPM1A mRNA, Western Blot was used to detect the expression ofPPM1A and Cleaved-caspase -3 protein, ELISA was used to analyze the secretion of TNF -α, IL -6 and IL -1α, and cell apoptosis was assessed by flow cytometry. Bioinformatics prediction and luciferase activity detection verified the targeted regulation of PPM1A by miR-93-5p. Results Compared with the NC group, the expression of miR-93-5p in the 16-HBEcells of the RSV group reduced, PPM1A mRNA, PPM1A protein, Cleaved-caspase-3 protein expression, TNF-α, IL-6, IL1α secretion and the rate of cell apoptosis increased. The Cleaved-caspase-3 protein level, TNF-α, IL-6, IL-1α secretion and apoptosis rate in 16-HBE of the miR-93-5p+RSV group were lower than those in the miR-NC+RSV group. miR-93-5ptargets and regulates the expression of PPM1A. Compared with the si-NC+RSV group, the Cleaved-caspase-3 protein level, the secretion of TNF-α, IL-6, IL-1α, and the rate of apoptosis in the 16-HBE of the si-PPM1A+RSV group were reduced, but the results in the pcDNA -PPM1A +RSV group were the opposite. Compared with the miR -93 -5p +pcDNA -NC +RSVgroup, the miR-93-5p+pcDNA-PPM1A+RSV group improved the Cleaved-caspase-3 protein level, TNF-α, IL-6, IL-1αsecretion and apoptosis rate of 16-HBE. The above differences were all statistically significant (all P<0.05). Conclusion MiR93-5p can inhibit RSV-infected bronchial epithelial cell apoptosis and inflammation by targeting PPM1A gene