微小RNA-93-5p调控Toll样受体4基因抑制呼吸道合胞病毒感染支气管上皮细胞炎症反应的机制研究

The Mechanism by Which miR-93-5p Regulates the TLR4 Gene to Inhibit the Inflammatory Response of Bronchial Epithelial Cells Infected by Respiratory Syncytial Virus

研究miR-93-5p调控TLR4基因抑制呼吸道合胞病毒(Respiratory syncytial sirus, RSV)感染支气管上皮细胞炎症反应。培养人支气管上皮细胞16-HBE,RSV感染,实验分为RSV+miR-NC组、RSV+miR-93-5p组、RSV+si-NC组、RSV+si-TLR4组、RSV+pcDNA-NC组、RSV+pcDNA-TLR4组、RSV+miR-93-5p+pcDNA-NC组、RSV+miR-93-5p+pcDNA-TLR4组。qRT-PCR评估miR-93-5p、TLR4 mRNA表达水平;酶联免疫吸附测定法(ELISA)检测TNF-α、IL-1β、IL-6水平;细胞计数试剂盒(CCK-8)法检测细胞增殖;Western blot检测CyclinD1、P21蛋白表达;双荧光素酶报告实验验证miR-93-5p对TLR4的靶向调控。与Con组比较,RSV组miR-93-5p表达水平显著降低,TLR4 mRNA表达水平显著增加(P<0.05)。与RSV+miR-NC组比较,RSV+miR-93-5p组16-HBE细胞中miR-93-5p表达水平显著增加,TNF-α、IL-1β、IL-6水平显著降低,细胞存活率、CyclinD1蛋白表达显著增加,P21蛋白表达显著降低(P<0.05)。与RSV+si-NC组比较,RSV+si-TLR4组16-HBE细胞中TLR4 mRNA表达水平显著降低,TNF-α、IL-1β、IL-6水平降低,细胞存活率、CyclinD1蛋白增加,P21蛋白减少(P<0.05)。与RSV+miR-93-5p+pcDNA-NC组比较,RSV+miR-93-5p+pcDNA-TLR4组16-HBE细胞中TLR4 mRNA表达水平显著增加,TNF-α、IL-1β、IL-6水平显著增加,细胞存活率、CyclinD1蛋白表达显著降低,P21蛋白表达显著增加(P<0.05)。miR-93-5p负调控TLR4基因抑制呼吸道合胞病毒感染支气管上皮细胞炎症反应。

This study investigated how miR-93-5p regulates the TLR4 gene in respiratory syncytial virus(RSV)infection of bronchial epithelial cells.Human 16-HBE bronchial epithelial cells were cultured and infected with RSV.Cells were divided into groups:RSV+miR-NC,RSV+miR-93-5p,RSV+si-NC,RSV+si-TLR4,RSV+pcDNA-NC,RSV+pcDNA-TLR4,RSV+miR-93-5p+pcDNA-NC,and RSV+miR-93-5p+pcDNA-TLR4.Real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to measure the expression levels of miR-93-5p and TLR4 mRNA;enzyme-linked immunosorbent assay was used to measure the levels of tumor necrosis factor-α(TNF-α)and interleukins(IL)-1βand-6;the Cell Counting Kit(CCK-8)method was used to detect cell proliferation;Western blot was used to measure cyclin 1(CyclinD1)and P21 protein expression;dual luciferase reporter assay was used to verify the targeted regulation of TLR4 by miR-93-5p.Compared with the control group,the expression level of miR-93-5p was significantly decreased in the RSV-infected group,and the expression level of TLR4 mRNA was significantly increased(P<0.05).Compared with the RSV+miR-NC group,the expression level of miR-93-5p in 16-HBE cells in the RSV+miR-93-5p group was significantly increased;the levels of TNF-α,IL-1β,and IL-6 were significantly decreased;the cell survival rate and CyclinD1 protein expression increased significantly;and P21 protein expression decreased significantly(P<0.05).Compared with the RSV+si-NC group,the expression level of TLR4 mRNA was significantly decreased in the 16-HBE cells of the RSV+si-TLR4 group;the levels of TNF-α,IL-1β,and IL-6 were significantly decreased;the cell survival rate and CyclinD1 protein expression were significantly increased;and the expression of P21 protein decreased significantly(P<0.05).Compared with the RSV+miR-93-5p+pcDNA-NC group,the expression level of TLR4 mRNA in the 16-HBE cells in the RSV+miR-93-5p+pcDNA-TLR4 group increased significantly;the levels of TNF-α,IL-1β,and IL-6 increased significantly;the cell survival rate and CyclinD1 protein expression decreased significantly;and P21 expression increased significantly(P<0.05).MiR-93-5p negatively regulates the TLR4 gene to inhibit the inflammatory response of bronchial epithelial cells infected by RSV.