摘要
目的:揭示miR-455-5p对呼吸道合胞病毒(RSV)感染致气道上皮细胞炎症反应的作用机制.方法:qRT-PCR检测30例健康体检儿童(健康组)、RSV感染轻症组(n=41)和重症组(n=31)患儿血清miR-455-5p水平.将16HBE细胞分为Control组、NC-agomir 组、miR-455-5p-agomir 组、NC-antagomir 组、miR-455-5p-antagomir 组.使用 Lipofectamine 3000 转染 16HBE 细胞后培养48h后,分为 Blank组、NC 组(转染了 NC-agomir 的细胞)、RSV+NC-agomir 组、RSV+miR-455-5p-agomir 组,用 RSV 病毒液感染 RSV+NC-agomir 组和 RSV+miR-455-5p-agomir 组 16HBE 细胞,Blank 组和 NC 组 16HBE 细胞正常培养.用 CCK-8 法和 EdU法检测细胞增殖、TUNEL法检测细胞凋亡、ELISA法检测上清液中TNF-α、IL-6、IL-8水平,qRT-PCR检测miR-455-5p和SOCS3的mRNA水平,Western blot检测SOCS3、IFN-α、STAT1、STAT2、p-STAT1和p-STAT2的蛋白水平.结果:与健康组相比,轻症组和重症组患儿的血清miR-455-5p水平降低(P＜0.05).与轻症组相比,重症组的血清miR-455-5p水平降低(P＜0.05).与Control组和NC-agomir组相比,miR-455-5p-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率升高(P＜0.05),TUNEL阳性率降低(P＜0.05),上清液中的TNF-α、IL-6和IL-8水平降低(P＜0.05),SOCS3 mRNA和蛋白水平降低(P＜0.05),IFN-α 蛋白、STAT1 和 STAT2 磷酸化水平升高(P＜0.05).与 Contro l 组和 NC-antagomir 组相比,miR-455-5p-antagomir 组 16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低(P＜0.05),TUNEL阳性率升高(P＜0.05),上清液中的TNF-α、IL-6和IL-8水平升高(P＜0.05),SOCS3 mRNA和蛋白水平升高(P＜0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平降低(P＜0.05).与Blank组和NC组相比,RSV+NC-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低(P＜0.05),TUNEL阳性率升高(P＜0.05),上清液中的TNF-α、IL-6和IL-8水平升高(P＜0.05),SOCS3 mRNA和蛋白水平升高(P＜0.05),IFN-α蛋白、STAT1 和 STAT2 磷酸化水平降低(P＜0.05).与 RSV+NC-agomir 组相比,RSV+miR-455-5p-agomir 组的 miR-455-5p 水平、相对细胞活力和EdU阳性率升高(P＜0.05),TUNEL阳性率降低(P＜0.05),上清液中的TNF-α、IL-6和IL-8水平降低(P＜0.05),SOCS3 mRNA和蛋白水平降低(P＜0.05),IFN-α蛋白、STAT1和STAT2磷酸化水平升高(P＜0.05).结论:miR-455-5p在RSV感染患儿血清中下调,上调miR-455-5p通过抑制SOCS3的转录和表达从而激活RSV感染的16HBE细胞中IFN-α介导的抗病毒反应.
Abstract
Objective:To investigate the effect of miR-455-5p on the inflammatory response of airway epithelial cells infected with respiratory syncytial virus(RSV)and its mechanism.Methods:The level of serum miR-455-5p was measured by qRT-PCR in 30 health children(health group),mild group(n=41)and severe group(n=31)children with RSV infection.16HBE cells were divided into Control group,NC-agomir group,miR-455-5p-agomir group,NC-antagomir group and miR-455-5p-antagomir group.16HBE cells were transfected by Lipofectamine 3000 and cultured for 48 h.Cell proliferation of 16HBE cells were detected by CCK-8 method and EdU method,and apoptosis was detected by TUNEL method.The levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-8 in the supernatant of 16HBE cells were detected by ELISA method.The mRNA levels of miR-455-5p and suppressor of cytokine signaling-3(SOCS3)were detected by qRT-PCR.The protein levels of SOCS3,interferon-α(IFN-α),signal transducer and activator of transcription(STAT)1,STAT2,p-STAT1 and p-STAT2 were detected by Western blot.16HBE cells were divided into Blank group,NC group(cells transfected with NC-agomir),RSV+NC-agomir group and RSV+miR-455-5p-agomir group.16HBE cells of RSV+NC-agomir group and RSV+miR-455-5p-agomir group were infected with RSV virus solution,and Blank group and NC group 16HBE cells were cultured nor-mally.Then,cell proliferation,apoptosis,inflammatory factors,miR-455-5p and SOCS3 mRNA levels,SOCS3,IFN-α,STAT1,STAT2,p-STAT1 and p-STAT2 protein levels were detected according to the above methods.Results:Compared with the health group,the level of serum miR-455-5p in mild group and severe group was lower than that in health group(P＜0.05).The level of serum miR-455-5p in se-vere group was lower than that in mild group(P＜0.05).Compared with Control group and NC-agomir group,miR-455-5p level,relative cell viability and EdU positive rate in miR-455-5p-agomir group increased(P＜0.05),TUNEL positive rate decreased(P＜0.05),TNF-α,IL-6 and IL-8 levels in supernatant decreased(P＜0.05),SOCS3 mRNA and protein levels decreased(P＜0.05),IFN-α protein,STAT1 and STAT2 phosphorylation increased(P＜0.05).Compared with Control group and NC-antagomir group,miR-455-5p level,relative cell via-bility and EdU positive rate in miR-455-5p-antagomir group decreased(P＜0.05),TUNEL positive rate increased(P＜0.05),TNF-α,IL-6 and IL-8 levels in supernatant increased(P＜0.05),SOCS3 mRNA and protein levels increased(P＜0.05),IFN-α protein,STAT1 and STAT2 phosphorylation decreased(P＜0.05).Compared with Blank group and NC group,miR-455-5p level,relative cell viability and E-dU positive rate in RSV+NC-agomir group decreased(P＜0.05),TUNEL positive rate increased(P＜0.05),TNF-α,IL-6 and IL-8 levels in supernatant increased(P＜0.05),SOCS3 mRNA and protein levels increased(P＜0.05),IFN-α protein,STAT1 and STAT2 phosphoryla-tion decreased(P＜0.05).Compared with RSV+NC-agomir group,miR-455-5p level,relative cell viability and EdU positive rate in RSV+miR-455-5p-agomir group increased(P＜0.05),TUNEL positive rate decreased(P＜0.05),TNF-α,IL-6 and IL-8 levels in super-natant decreased(P＜0.05),SOCS3 mRNA and protein levels decreased(P＜0.05),IFN-α protein,STAT1 and STAT2 phosphorylation levels increased(P＜0.05).Conclusion:miR-455-5p is down-regulated in the serum of RSV-infected children,and up-regulated miR-455-5p activates IFN-α-mediated antiviral response in RSV-infected 16HBE cells by inhibiting SOCS3 transcription and expression.
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