miR-15a-5p调控Wnt通路在百草枯致肺纤维化中的机制  

Mechanism of miR-15a-5p regulation of Wnt pathway in paraquat-induced pulmonary fibrosis

作　　者：王静 姬晓航 王蒙蒙 张炜 丁伟超 陈娟 冯靖 孟健康 孙兆瑞 聂时南 Wang Jing;Ji Xiaohang;Wang Mengmeng;Zhang Wei;Ding Weichao;Chen Juan;Feng Jing;Meng Jiankang;Sun Zhaorui;Nie Shinan(Department of Emergency Medicine,Jinling Hospital(General Hospital of Eastern Theater Command),Medical School of Nanjing University,Nanjing 210002,China;Department of experiment center,School of Medicine Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]南京大学医学院附属金陵医院(中国人民解放军东部战区总医院)急诊医学科,南京210002 [2]江苏大学医学院实验中心,镇江212013

出　　处：《中华急诊医学杂志》2024年第8期1128-1133,共6页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金面上项目(82172182)。

摘　　要：目的探讨miR-15a-5p调控Wnt信号通路对PQ致肺纤维化的影响及分子机制。方法构建PQ诱导的16HBE细胞模型,采用高通量miRNA芯片技术和RT-qPCR筛选表达差异明显的miR-15a-5p进行实验。实验分组为:NC组(对照组):无特殊处理;PQ组:50μmol/L PQ处理细胞72 h;miR-15a-5p组:转染miR-15a-5p过表达慢病毒的16HBE稳转株;miR-15a-5p+PQ组:50μmol/L PQ处理稳转株细胞72 h。RT-qPCR和Western blot检测Wnt通路相关基因Wnt3α和β-catenin、成纤维细胞标记基因Collagen I、Vimentin和αSMA,上皮细胞标记基因Occludin和CK18表达情况。构建PQ诱导的肺纤维化小鼠模型,采用Western blot、HE染色和免疫组织化学检测蛋白表达及肺组织损伤情况。数据以均数±标准差(x±s)表示,采用独立样本t检验分析两组间数据。结果细胞损伤模型中,Wnt3α、β-catenin、成纤维细胞标记基因Collagen I、Vimentin和αSMA表达显著上调(P<0.05),上皮细胞标记基因Occludin和CK18显著下调(P<0.05),过表达miR-15a-5p可靶向抑制Wnt3α表达并缓解PQ诱导的EMT进程。动物模型中,Wnt3α、β-catenin、成纤维细胞标记基因Collagen I、Vimentin和αSMA蛋白水平显著升高(P<0.01),肺组织结构紊乱并发生纤维化,过表达miR-15a-5p可抑制Wnt3α蛋白表达水平(P<0.05)且改善肺组织损伤。结论miR-15a-5p可通过调控Wnt3α/β-catenin信号通路改善PQ导致的肺损伤,从而抑制肺纤维化的发生发展。Objective To investigate the effect and molecular mechanism of miR-15a-5p regulation Wnt signaling pathway in PQ-induced pulmonary fibrosis.Methods The PQ-induced 16HBE cell model was constructed,high-throughput miRNA chip and RT-qPCR were used to screen for miR-15a-5p with significant differences.The experimental groups were as follows:NC group(normal control);no special treatment;PQ group:50μmol/L PQ treated cells for 72 h;miR-15a-5p group:16HBE stable cell lines transfected with miR-15a-5p overexpressing lentivirus;miR-15a-5p+PQ group:Stable cell lines were treated with 50μmol/L PQ for 72 h.The expression of Wnt pathway-related genes Wnt3αandβ-catenin,fibroblast marker genes Collagen I,Vimentin andαSMA,epithelial marker genes Occludin and CK18 were detected by RT-qPCR and Western blot.The mice model of PQ-induced pulmonary fibrosis was constructed,and the protein expression and lung tissue injury were detected by Western blot,HE staining and immunohistochemistry.Data were expressed as mean±standard deviation,and independent sample t-test was used to analyze the data between the two groups.Results The expressions of Wnt3α,β-catenin,fibroblast marker genes Collagen I,Vimentin andαSMA significantly up-regulated in cell injury models(P<0.05),the epithelial cell marker genes Occludin and CK18 significantly down-regulated(P<0.05),overexpression of miR-15a-5p could inhibit the expression of Wnt3αand alleviated the EMT induced by PQ.In animal models,Wnt3α,β-catenin,fibroblast marker genes Collagen I,Vimentin andαSMA significantly increased(P<0.01),the structure of lung tissue was disordered and fibrosis occurred,overexpression of miR-15a-5p inhibited the expression of Wnt3αprotein(P<0.05)and ameliorated lung tissue injury.Conclusions miR-15a-5p ameliorates PQ-induced lung injury by modulating the Wnt3α/β-catenin signaling pathway,thereby inhibiting the development of pulmonary fibrosis.

关 键 词：miRNA miR-15a-5p 百草枯 肺纤维化 16HBE细胞 Wnt信号通路 Wnt3α β-catenin EMT 

分 类 号：R563[医药卫生—呼吸系统]