温石棉暴露诱发核糖体DNA拷贝数变异及DNA损伤反应研究

Effect of exposure to chrysotile on ribosomal DNA copy number variation and DNA damage response

目的探索温石棉暴露对核糖体DNA(rDNA)拷贝数的影响及其与DNA损伤反应的关系,为石棉致癌机制研究提供依据。方法分别用1.25、2.5、5μg/cm^(2)(低、中、高浓度)的温石棉悬液处理MeT-5A人胸膜间皮细胞,磷酸盐缓冲液处理作为对照。分别收集处理6、24、48和72 h的细胞,采用实时荧光定量PCR检测rDNA拷贝数及核仁蛋白基因BIRC5、HRAS、GINS4和RRM2 mRNA表达;使用Muse细胞分析仪及相应试剂盒检测细胞凋亡和DNA损伤。比较不同处理时间不同温石棉浓度组细胞rDNA拷贝数、DNA损伤反应和核仁蛋白基因mRNA表达水平。结果温石棉悬液处理6、48、72 h时,低、中、高浓度组和对照组45S rDNA拷贝数差异均有统计学意义(P<0.05);处理6 h时,各浓度组45S rDNA拷贝数均低于对照组(P<0.05);处理48、72 h时,高浓度组45S rDNA拷贝数高于低、中浓度组(P<0.05)。处理24、48、72 h时,各组5S rDNA拷贝数比较,差异均有统计学意义(P<0.05);处理24、48 h时,中、高浓度组5S rDNA拷贝数均低于对照组(P<0.05);处理24、72 h时,中、高浓度组5S rDNA拷贝数均低于低浓度组(P<0.05)。不同处理时间点各组总细胞凋亡率比较,差异均有统计学意义(P<0.05);中、高浓度组总细胞凋亡率均高于对照组(P<0.05),以晚期凋亡为主。处理72 h时,各组ATM激活率和DNA双链断裂率比较,差异均有统计学意义(P<0.05);中、高浓度组细胞ATM激活率和DNA双链断裂率均高于对照组(P<0.05)。处理24、48 h时,各组BIRC5、HRAS、GINS4和RRM2 mRNA相对表达量比较,差异均有统计学意义(P<0.05);中、高浓度组BIRC5、HRAS、GINS4和RRM2 mRNA相对表达量均低于对照组(P<0.05)。结论温石棉暴露可诱发人胸膜间皮细胞rDNA拷贝数变异,引起相关核仁蛋白表达改变,可能参与DNA损伤反应过程的调控。

Objective To investigate the effect of chrysotile exposure on ribosomal DNA(rDNA)copy number and DNA damage response,so as to provide insights into the mechanism of asbestos-induced carcinogenesis.Methods Human pleural mesothelial MeT-5A cells were treated with chrysotile suspensions at doses of 1.25,2.5 and 5μg/cm^(2)(low-,medium-,high-dose group),while PBS served as controls.MeT-5A cells were harvested 6,24,48 and 72 h post-treat⁃ment,and the rDNA copy numbers and the BIRC5,HRAS,GINS4 and RRM2 mRNA expression were determined using a quantitative real-time PCR(qPCR)assay.The apoptosis of MeT-5A cells and DNA damage were detected using Muse cell analyzer.The rDNA copy numbers,DNA damage responses and BIRC5,HRAS,GINS4 and RRM2 mRNA ex⁃pression were compared in MeT-5A cells treated with different doses of chrysotile suspensions.Results There were sig⁃nificant differences in 45S rDNA copy numbers among low-,medium-,high-dose groups and the control groups 6,48 and 72 h post-treatment with chrysotile suspensions,and significantly lower 45S rDNA copy numbers were measured in low-,medium-and high-dose groups than in the control group 6 h post-treatment,while significantly higher 45S rDNA copy numbers were found in the high-dose group than in low-and medium-dose groups 48 and 72 h post-treatment(all P<0.05).There were significant differences in 5S rDNA copy numbers among low-,medium-,high-dose groups and the control groups 24,48 and 72 h post-treatment with chrysotile suspensions,and significantly lower 5S rDNA copy numbers were measured in medium-and high-dose groups than in the control group 24 and 48 h post-treatment,while significantly lower 5S rDNA copy numbers were found in medium-and high-dose groups than in the low-dose group 24,72 h post-treatment(all P<0.05).There were significant differences in the overall apoptotic rate of MeT-5A cells among groups at different time points,and the overall apoptotic rate of MeT-5A cells were significantly higher in medium-and high-dose groups than in the control group(all P<0.05),with late-stage apoptosis predominantly detected.There were significant differences in the rates of ATM activation and DNA double-strand break in MeT-5A cells among groups 72 h post-treatment,and higher rates of ATM activation and DNA double-strand break were measured in medium-and high-dose groups than in the control group(all P<0.05).In addition,there were significant differences in the relative mRNA expression of BIRC5,HRAS,GINS4 and RRM2 genes among groups 24 and 48 h post-treatment,and significantly lower BIRC5,HRAS,GINS4 and RRM2 mRNA expression was quantified in medium-and high-dose groups than in the control group(all P<0.05).Conclusion Exposure to chrysotile may induce rDNA copy number varia⁃tions and altered expression of nucleolar proteins in human pleural mesothelial cells,which may be involved in the reg⁃ulation of DNA damage responses.