| 黄 娜,王宝岗,姜亚丽,张 垚,汤丽萍.miR-455-5p靶向SOCS3抑制RSV感染致气道上皮炎症反应的机制研究[J].,2024,(9):1614-1622 |
| miR-455-5p靶向SOCS3抑制RSV感染致气道上皮炎症反应的机制研究 |
| miR-455-5p Attenuates Inflammation of Airway Epithelial Cells Infected by Respiratory Syncytial Virus by Inhibiting SOCS3 |
| 投稿时间：2023-12-24 修订日期：2024-01-29 |
| DOI：10.13241/j.cnki.pmb.2024.09.003 |
| 中文关键词: miR-455-5p 细胞因子信号传导抑制因子3 呼吸道合胞病毒 气道上皮细胞 炎症反应 |
| 英文关键词: miR-455-5p Cytokine signal transduction inhibitor 3 Respiratory syncytial virus Airway epithelial cells Inflammatory response |
| 基金项目:国家自然科学基金项目（82000644） |
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| 中文摘要: |
| 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48 h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Control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。 |
| 英文摘要: |
| 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 normally. 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 severe 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 viability 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 EdU 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 phosphorylation 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 supernatant 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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