长链非编码RNA-PVT1介导JAK/STAT3信号通路对肺纤维化的研究  

Long non-coding RNA-PVT1 induces the progression of pulmonary fibrosis via the JAK/STAT3 signaling pathway

作　　者：卓超洲 沈观乐 雷朝君 余瑞林 梁杰 高妩媚 魏艾 Zhuo Chaozhou;Shen Guanle;Lei Chaojun;Yu Ruilin;Liang Jie;Gao Wumei;Wei Ai(Department of Respiratory Medicine,Shenzhen Longhua District People’s Hospital,Shenzhen 518109,China)

机构地区：[1]深圳市龙华区人民医院呼吸内科,深圳518109

出　　处：《新医学》2022年第7期496-502,共7页Journal of New Medicine

基　　金：深圳市基础研究资助项目(JCYJ20180228164139811)。

摘　　要：目的探讨长链非编码RNA-浆细胞瘤变异易位基因1/蛋白质酪氨酸激酶/信号转导和转录激活因子3(lnc-PVT1/JAK/STAT3)信号通路参与肺纤维化的作用机制。方法采用不同浓度脂多糖(LPS)作用于人正常肺上皮细胞BEAS-2B,用CCK-8法检测细胞活性、蛋白免疫印迹法检测α-SMA的蛋白相对表达量,对LPS药物浓度进行筛选,选取LPS刺激后最适浓度的BEAS-2B建立肺纤维化体外模型;体外合成针对lnc-PVT1的3个小干扰RNA(siRNA)抑制靶点及对照siRNA,瞬时转染到BEAS-2B,用定量逆转录PCR(RT-qPCR)验证lnc-PVT1的表达,选取有效抑制靶点用于后续研究。实验分为空白对照(Con)组、LPS组、LPS+siRNA对照(LPS+siNC)组和LPS+siPVT1组。瞬时转染siPVT1及siRNA对照进入BEAS-2B后,用10μg/mL LPS刺激24 h,用CCK-8法检测细胞活力,划痕检测细胞迁移,RT-qPCR检测lnc-PVT1 mRNA相对表达量,ELISA检测细胞上清液中IL-6、IL-1β和TNF-α含量,蛋白免疫印迹法检测细胞中α-平滑肌肌动蛋白(α-SMA)、STAT3和p-STAT3蛋白表达。结果与Con组相比,10μg/mL LPS处理BEAS-2B细胞后细胞增殖能力增强,α-SMA蛋白相对表达量也上调(P均<0.01);与LPS组相比,敲低lnc-PVT1可抑制LPS诱导的BEAS-2B细胞增殖和细胞迁移(P均<0.01),降低IL-6、IL-1β和TNF-α等炎症因子的含量(P均<0.01),降低α-SMA的mRNA和蛋白表达及磷酸化STAT3蛋白相对表达量(P均<0.01),而STAT3蛋白相对表达量无明显变化(P>0.05)。结论敲低lnc-PVT1可改善LPS诱导的BEAS-2B细胞纤维化过程,可能与lnc-PVT1调控JAK/STAT3信号通路有关。Objective To investigate the role of long non-coding RNA-PVT1/JAK/STAT3 signaling pathway in pulmonary fibrosis.Methods BEAS-2B cells were treated with different concentrations of LPS.Cell viability was assessed by CCK-8.The relative expression level ofα-SMA protein was determined by western blot.The concentration of LPS was screened.BEAS-2B cells were treated with the optimal concentration of LPS to establish the pulmonary fibrosis model in vitro.BEAS-2B cells were transiently transfected with lnc-PVT1 siRNA and control siRNA followed by LPS treatment.The expression level of lnc-PVT1 was determined by RT-qPCR.Effective inhibitory targets were selected for subsequent experiment.BEAS-2B cells were divided into the control(Con)group,LPS group,LPS+siRNA control(LPS+siNC)group and LPS+siPVT1 group.After transiently transfected with siPVT1 and siRNA control,BEAS-2B cells were treated with 10μg/mL LPS for 24 h.Cell viability was assessed by CCK-8.Cell migration was evaluated by cell scratch test.The relative expression level of lnc-PVT1 mRNA was measured by RT-qPCR.The expression levels of IL-6,IL-1βand TNF-αin the cultured supernatant were measured by ELISA.The expression levels ofα-SMA,STAT3 and p-STAT3 were assessed by western blot.Results In the LPS group treated with 10μg/mL LPS for 24 h,the cell proliferation was significantly higher and the expression level of fibrotic markerα-SMA protein was remarkably up-regulated than those in the Con group(both P<0.01).Compared with the LPS group,knockdown of lnc-PVT1 could significantly inhibit the cell proliferation and migration induced by LPS(both P<0.01),down-regulate the expression levels of IL-6,IL-1βand TNF-α(all P<0.01),and decrease the expression levels ofα-SMA mRNA and protein as well as the phosphorylated STAT3 protein(all P<0.01).However,the relative expression level of total STAT3 protein was not affected.Conclusion Knockdown of lnc-PVT1 can effectively attenuate the LPS-induced BEAS-2B cell fibrosis,which may be associated with the role of lnc-PVT1 i

关 键 词：浆细胞瘤变异易位基因1 肺纤维化 蛋白质酪氨酸激酶/信号转导和转录激活因子3 炎症因子 

分 类 号：R563[医药卫生—呼吸系统]