Li R, Yu W K, Wang Q, Zhu L J, Zhang F F
School of Public Health, Hangzhou Medical College, Hangzhou 310000, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2024 Sep 20;42(9):668-672. doi: 10.3760/cma.j.cn121094-20240112-00014.
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. 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- test. 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 (<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 (<0.001), which was consistent with the results of chip analysis. After exposure of Met-5A cells, it was found that compared with the control group, the expression levels of Vimentin and Twist genes, hsa-miR-155-5p, hsa-miR-34b-5p and hsa-miR-34c-5p in the crocidolite group were increased, while the expression level of E-cadherin gene was decreased (<0.05). Compared with the control group, the expression levels of Vimentin, Twist and E-cadherin genes in chrysotile group were increased, while the expression levels of hsa-miR-34b-5p, hsa-miR-34c-5p and hsa-miR-28-5p were decreased (<0.05) . Long-term exposure to chrysotile and crocidolite could cause Met-5A cells to produce miRNAs and EMT-related gene expression changes similar to mesothelioma cells.
研究长期暴露于温石棉和青石棉对人胸膜间皮细胞中微小RNA（miRNA）及上皮-间质转化（EMT）相关基因表达的影响。2020年11月，采用荧光定量聚合酶链反应（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。从课题组前期的miRNA芯片数据中筛选出人胸膜间皮瘤细胞中表达异常的miRNA，并利用miRWalk数据库（http://mirwalk.umm.uni-heidelberg.de）预测差异表达miRNA的靶基因。采用RT-qPCR验证细胞系中EMT相关miRNA的异常表达。将Met-5A细胞分别用5μg/cm²的温石棉和青石棉处理，每次处理48小时，每周1次，共处理10次。设置温石棉组、青石棉组和对照组，对照组加入等量的PBS。采用RT-qPCR检测各组EMT相关基因的表达变化及异常表达的miRNA。组间差异采用单因素方差分析，对照组与实验组之间的差异采用Dunnett检验。与Met-5A细胞相比，NCl-H2052细胞和NCl-H2452细胞中波形蛋白（Vimentin）和Twist基因的表达水平升高，E-钙黏蛋白（E-cadherin）基因的表达水平降低（<0.001）。预测了miRNA芯片中表达异常的miRNA的靶基因，结果显示有4种与EMT相关的异常表达miRNA，并在细胞系中验证了这4种miRNA的表达。与Met-5A细胞相比，NCl-H2052细胞和NCl-H2452细胞中hsa-miR-155-5p的表达水平升高，hsa-miR-34b-5p、hsa-miR-34c-5p和hsa-miR-28-5p的表达水平降低（<0.001），这与芯片分析结果一致。Met-5A细胞暴露后发现，与对照组相比，青石棉组中Vimentin和Twist基因、hsa-miR-155-5p、hsa-miR-34b-5p和hsa-miR-34c-5p的表达水平升高，而E-cadherin基因的表达水平降低（<0.05）。与对照组相比，温石棉组中Vimentin、Twist和E-cadherin基因的表达水平升高，而hsa-miR-34b-5p、hsa-miR-34c-5p和hsa-miR-28-5p的表达水平降低（<0.05）。长期暴露于温石棉和青石棉可导致Met-5A细胞产生与间皮瘤细胞相似的miRNA及EMT相关基因表达变化。
