呼吸道合胞病毒通过调控miR-409-3p/SIRT1通路促进支气管上皮细胞凋亡与炎症因子表达

Respiratory syncytial virus promotes apoptosis and expression of inflammatory factors in bronchial epithelial cells by regulating miR-409-3p/SIRT1 pathway

目的呼吸道合胞病毒(respiratory syncytial virus, RSV)对人支气管上皮细胞(human bronchial epithelial cells, HBECs)凋亡和炎性因子表达的影响及可能机制。方法体外培养HBECs,分别转染miR-409-3p抑制剂、SIRT1过表达载体或共转染miR-409-3p抑制剂与SIRT1小干扰RNA后,采用RSV感染进行转染,然后RT-qPCR法检测细胞中miR-409-3p和SIRT1 mRNA表达水平,CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡率,Western Blot检测细胞中Ki-67、p21、Bcl-2和Bax蛋白表达,试剂盒法检测细胞培养上清液中IL-6和TNF-α表达水平。双荧光素酶报告基因实验验证SIRT1和miR-409-3p调控关系。结果 RSV感染促进HBECs细胞中miR-409-3p的表达(P<0.05),而抑制SIRT1表达(P<0.05),降低HBECs细胞增殖活性及细胞中Ki-67和Bcl2的蛋白表达(P<0.05),促进HBECs凋亡及细胞中p21和Bax的蛋白及IL-6和TNF-α表达(P<0.05)。沉默miR-409-3p或过表达SIRT1可提高RSV感染的HBECs增殖活性及细胞中Ki-67和Bcl2的蛋白表达(P<0.05),而抑制细胞凋亡率、p21和Bax的蛋白及IL-6和TNF-α表达(P<0.05)。miR-409-3p靶向负调控SIRT1表达。沉默SIRT1逆转了沉默miR-409-3p对RSV感染的HBECs增殖、凋亡及炎性因子IL-6和TNF-α表达的影响(P<0.05)。结论呼吸道合胞病毒通过调控miR-409-3p/SIRT1通路促进支气管上皮细胞凋亡与炎症因子表达。

Objective To explore the effect of respiratory syncytial virus(RSV) on the apoptosis and the expression of inflammatory factors in human bronchial epithelial cells(HBECs), and investigate its possible mechanisms. Methods In vitro cultured HBECs were transfected with miR-409-3 p inhibitor, overexpression vector of silent information regulator 1(SIRT1) along, or co-transfected with miR-409-3 p inhibitor and SIRT1 small interfering RNA(siRNA), respectively. After RSV infection, RT-qPCR was used to detect the expression levels of miR-409-3 p and SIRT1 in the transfected cells. Cell counting kit-8(CCK-8) assay and flow cytometry were performed to determine cell proliferation as well as cell apoptosis. Western blotting was also adopted to verify the expression of Ki-67, p21, Bcl-2 and Bax. In addition, corresponding reagent testing kits were used to detect the contents of IL-6 and TNF-α in culture supernatant. Finally, the regulatory relationship between SIRT1 and miR-409-3 p was verified by the dual luciferase reporter assay. Results RSV infection promoted the expression of miR-409-3 p(P<0.05), inhibited that of SIRT1(P<0.05), decreased cell proliferation and protein expression of Ki-67 and Bcl2(P<0.05), and promoted cell apoptosis and protein expression of p21 and Bax and contents of IL-6 and TNF-α(P<0.05) in HBECs cells. Silencing miR-409-3 p or overexpressing SIRT1 resulted in the enhanced the proliferation activity and the protein expression of Ki-67 and Bcl2 in RSV-infected HBECs cells(P<0.05), but decreased apoptotic rate, protein expression of p21 and Bax, and contents of IL-6 and TNF-α(P<0.05). miR-409-3 p showed a negatively targeted regulation to SIRT1 expression. Silencing SIRT1 reversed the effects of silenced miR-409-3 p on the proliferation and apoptosis of RSV-infected HBECs as well as on the expression of inflammatory factors IL-6 and TNF-α(P<0.05). Conclusion RSV promotes the apoptosis and the expression of inflammatory factors in bronchial epithelial cells by regulating miR-409-3 p/SIRT1 pathway.