石棉长期暴露致人间皮细胞miRNAs及上皮间充质转化相关基因表达改变  

Expression changes of miRNAs and EMT-related genes in human mesothelial cells induced by long-term exposure to asbestos

作　　者：李芮 余温珂 王旗 朱丽瑾 张芳芳 Li Rui;Yu Wenke;Wang Qi;Zhu Lijin;Zhang Fangfang(School of Public Health,Hangzhou Medical College,Hangzhou 310000,China)

机构地区：[1]杭州医学院公共卫生学院,杭州310000

出　　处：《中华劳动卫生职业病杂志》2024年第9期668-672,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：浙江省自然科学基金(LQY18H260001、LGD21C040008);杭州医学院院所专项计划项目(YS2022012)。

摘　　要：目的探讨温石棉和青石棉长期暴露对人胸膜间皮细胞miRNAs和上皮间充质转化(EMT)相关基因表达的影响。方法于2020年11月,应用荧光定量聚合酶链式反应技术(RT-qPCR)检测人胸膜间皮瘤细胞(NCl-H2052细胞、NCl-H2452细胞)和人正常间皮细胞(Met-5A细胞)中EMT相关基因的表达;从课题组前期miRNA芯片数据中筛选出在人胸膜间皮瘤细胞中异常表达的miRNAs,利用miRWalk数据库(http://mirwalk.umm.uni-heidelberg.de)对差异表达的miRNAs进行靶基因预测,应用RT-qPCR对与EMT相关的异常表达的miRNAs在细胞系中进行验证;分别用5μg/cm 2的温石棉和青石棉对Met-5A细胞进行染毒,每周一次,每次48 h,共处理10次,分别设置为温石棉组,青石棉组,同步设置对照组,加入同等体积的PBS。应用RT-qPCR检测各组细胞EMT相关基因和异常表达miRNAs的表达变化,各组间差异用单因素方差分析进行比较,对照组与各实验组的差异用Dunnet-t检验进行比较。结果与Met-5A细胞比较,NCl-H2052细胞和NCl-H2452细胞的波形蛋白(Vimentin)、细胞转录因子(Twist)基因表达水平升高,钙黏蛋白(E-cadherin)基因表达水平降低(P<0.001);对miRNA芯片结果中异常表达的miRNAs进行靶基因预测,结果显示,与EMT相关的异常表达miRNAs有4个,在细胞系中验证这4个miRNAs的表达,与Met-5A细胞比较,NCl-H2052细胞和NCl-H2452细胞的hsa-miR-155-5p表达水平升高,hsa-miR-34b-5p、hsa-miR-34c-5p和hsa-miR-28-5p表达水平降低(P<0.001),该结果与芯片分析结果一致。对Met-5A细胞进行染毒发现,与对照组比较,青石棉组Vimentin和Twist基因、hsa-miR-155-5p、hsa-miR-34b-5p、hsa-miR-34c-5p的表达水平升高,E-cadherin基因表达水平降低(P<0.05);与对照组比较,温石棉组Vimentin、Twist和E-cadherin基因的表达水平升高,hsa-miR-34b-5p、hsa-miR-34c-5p、hsa-miR-28-5p的表达水平降低(P<0.05)。结论温石棉与青石棉长期暴露均可引起Met-5A细胞产生与间皮瘤细胞Objective To investigate the effects of long-term exposure to chrysotile and crocidolite on miRNAs and epithelial mesenchymal transformation(EMT)-related gene expression in human pleural mesothelial cells.Methods In November 2020,fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expressions of EMT-related genes in human pleural mesothelioma cells(NCl-H2052 cells,NCl-H2452 cells)and human normal mesothelial cells(Met-5A cells).MiRNAs with abnormal expression in human pleural mesothelioma cells were screened out from the previous miRNA chip data of research group,and target genes of differentially expressed miRNAs were predicted using miRWalk database(http://mirwalk.umm.uni-heidelberg.de).RT-qPCR was used to verify the abnormal expression of EMT-related miRNAs in cell lines.Met-5A cells were treated with 5μg/cm 2 chrysotile and crocidolite respectively for 48 h a time,once a week and a total of 10 times.Chrysotile group,crocidolite group and control group were set up.And the control group was added with the same volume of PBS.The expression changes of EMT-related genes and abnormal expression miRNAs in each group were detected by RT-qPCR.The differences among the groups were compared by one-way ANOVA,and the differences between the control group and the experimental group were compared by dunnet-t test.Results Compared with Met-5A cells,the expression levels of Vimentin and Twist genes were increased,and the expression level of E-cadherin genes was decreased in NCl-H2052 cells and NCl-H2452 cells(P<0.001).Target genes of miRNAs with abnormal expression in miRNA chip were predicted,and the results showed four abnormally expressed miRNAs associated with EMT and verified the expression of these four miRNAs in the cell lines.Compared with Met-5A cells,the expression level of hsa-miR-155-5p was increased in NCl-H2052 cells and NCl-H2452 cells,the expression levels of hsa-miR-34b-5p,hsa-miR-34c-5p and hsa-miR-28-5p were decreased in NCl-H2052 cells and NCl-H2452 cells(P<0.001),which was

关 键 词：间皮瘤 青石棉 温石棉 恶性胸膜间皮瘤 上皮间充质转化 MIRNAS 

分 类 号：R114[医药卫生—卫生毒理学]