MEHP对小鼠睾丸间质细胞DNA甲基化水平和细胞周期进程的影响

Effect of MEHP on DNA Methylation Level and Cell Cycle Progression of Mouse Leydig Cells

目的探讨邻苯二甲酸单(2-乙基已基)酯(MEHP)对小鼠睾丸间质细胞(TM-3)DNA甲基化水平和周期进程的影响。方法体外培养TM-3细胞,MEHP按照0、200、400、800μmol·L^(-1)共4个梯度进行分组,待其长到对数生长期,染毒24 h后处理细胞;采用Western blot检测DNMT1、DNMT3B两种甲基化转移酶(DNMTs)和转录因子FOXO1蛋白表达水平,流式细胞术检测细胞周期进程,RT-qPCR技术检测细胞周期蛋白依赖性激酶CDK2、细胞周期抑制因子P27 mRNA表达水平;给予TM-3细胞不同浓度的甲基化抑制剂5-氮杂脱氧胞苷(5-Aza-Cdr)溶液(0、10^(-4)、10^(-3)、10^(-2)、10^(-1)、10μmol·L^(-1))处理,采用CCK-8法检测细胞存活率,确定干预组染毒剂量;设置对照组(0μmol·L^(-1))、400μmol·L^(-1)MEHP染毒组、MEHP(400μmol·L^(-1))+5-Aza-Cdr(10-4μmol·L^(-1))干预组,采用CCK-8法、Western blot、流式细胞术、RT-qPCR法检测各项实验指标。结果Western blot结果显示,与0μmol·L^(-1)组相比,400、800μmol·L^(-1)MEHP染毒组DNMT1蛋白表达水平降低,DNMT3B蛋白表达水平升高(P均<0.01);200、400、800μmol·L^(-1)组FOXO1蛋白表达水平均升高(P均<0.01)。流式细胞术结果显示,MEHP染毒导致细胞增殖受阻。RT-qPCR结果显示,与对照组相比,随着染毒剂量的增加,P27 mRNA表达水平升高,CDK2 mRNA表达水平降低(P均<0.05);抑制剂干预后Western blot结果显示,与400μmol·L^(-1)MEHP染毒组相比,MEHP+5-Aza-Cdr干预组DNMT1、DNMT3B蛋白表达水平降低,FOXO1蛋白表达水平升高(P均<0.05);RT-qPCR结果显示,与400μmol·L^(-1)MEHP染毒组相比,MEHP+5-Aza-Cdr干预组P27 mRNA表达水平上升(P<0.05);流式细胞术结果显示,与对照组相比,400μmol·L^(-1)MEHP染毒组、MEHP+5-Aza-Cdr干预组细胞均阻滞在G1期(P均<0.01)。结论MEHP影响TM-3细胞正常分裂与增殖,与DNMTs相关蛋白表达水平改变、甲基化水平的异常修饰有关。

Objective To investigate the effect of phthalic acid mono-2-ethylhexyl ester(MEHP)on DNA methylation level and cycle progression of mouse Leydig cells(TM-3).Methods TM-3 cells were cultured in vitro,MEHP was grouped according to the four gradients of 0,200,400 and 800μmol·L^(-1).When the cells grew to the logarithmic growth phase,they were treated after 24 h of exposure.Western blot was used to detect the expression levels of DNMT1 and DNMT3B methylation transferases(DNMTs)and transcription factor FOXO1.Cell cycle progression was detected by flow cytometry;RT-qPCR was used to detect the mRNA expression of cyclin-dependent kinase CDK2 and cell cycle inhibitor P27.Treatment of TM-3 cells with different concentrations of 5-Aza-Cdr solution(0,10^(-4),10^(-3),10^(-2),10-1,10μmol L^(-1))at different concentrations of TM-3 cells,Cell viability was determined by the CCK-8 assay,the toxic dose of the intervention group was determined;The control group(0μmol·L^(-1)),400μmol L^(-1)MEHP infected group,MEHP(400μmol·L^(-1))+5-Aza-Cdr(10-4μmol·L^(-1))intervention group were set.These experimental indexes were measured by CCK-8,Western blot,flow cytometry,and RT-qPCR.Results The results of Western blot showed that compared with the 0μmol·L^(-1)group,the expression of DNMT1 protein in 400 and 800μmol·L^(-1)groups decreased,but the expression level of DNMT3B protein increased(P all<0.01).200,400 and 800μmol·L^(-1)groups FOXO1 protein expression level increased(P all<0.01).The flow cytometry results showed that MEHP infection caused cell proliferation blocked.The results of RT-qPCR showed that compared with the control group,with the increase of exposure doses,the expression levels of P27 mRNA increased,and the expression level of CDK2 mRNA decreased(P all<0.05).After inhibitor intervention,the Western blot results showed that compared with 400μmol·L^(-1)MEHP exposure group,the protein expression levels of DNMT1 and DNMT3B in MEHP+5-Aza-Cdr intervention group decreased,and the protein expression level of FOXO1 increased(P all<0.05),and RT-qPCR results showed that compared with the 400μmol·L^(-1)MEHP exposure group,the expression level of P27 mRNA in the MEHP+5-Aza-Cdr intervention group was increased(P<0.05).The flow cytometry results showed that compared with the control group,the cells in the 400μmol·L^(-1)MEHP exposure group and MEHP+5-Aza-Cdr intervention group were arrested in G1 stage(P all<0.01).Conclusion MEHP can affect TM-3 cell division and proliferation which related to the change of DNMTs-related protein expression level and abnormal modification of total methylation level.