聚-ADP-核糖基化在六价铬诱导人支气管上皮细胞基因组DNA甲基化水平改变中的作用

摘要

Objective To reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (Ⅵ),and to discuss the relations between them.Methods The pre-established Poly (ADP-ribose) glycohydrolase (PARG) deficient cells and 16HBE cells were treated with different concentrations of Cr (Ⅵ),and the changes of total genomic DNA methylation level in different groups were detected by methylation immunofluorescent detection,as well as the changes of the activity of methyltransferases.Moreover,RT-PCR and western blotting method were applied to analyze the changes of expression of DNMT1,DNMT3a,DNMT3b and MBD2,upon the protein level.Results After treated by Cr(Ⅵ) for 24 h,the healthy 16HBE cells showed a significant lower level of genomic DNA methylation;however,there was no significant changes (P ＞ 0.05) found in PARG deficient cells by immunofluorescence assay.When the dose of Cr(Ⅵ) reached 5.0 μmol/L,the activity of methyltransferases in 16HBE cells and PARG deficient cells(49.33 ± 2.65,80.05 ± 2.05) decreased by 20％ and 50％ comparing with contrast group (99.27 ± 1.10,99.30 ± 0.60).After treated by Cr(Ⅵ) for 24 h,the expression of mRNA and protein level among DNMT1,DNMT3a,DNMT3b and MBD2 decreased significantly in healthy 16HBE cells; and the expression of DNMT1 and DNMT3a decreased in PARG deficiency cells.The relevant expression levels of mRNA of DNMT1 were separately (0.99 ±0.09),(0.79 ±0.10),(0.59 ±0.13) and (0.39 ± 0.02) (F =247.17,P ＜ 0.01),the expression levels of protein were separately (1.00 ± 0.03),(0.69±0.15),(0.65±0.10) and (0.55 ±0.13) (F=214.12,P＜0.01),the expression levels of DNMT3a mRNA were separately (1.00 ±0.04),(0.93 ±0.11),(0.79 ±0.07),(0.59 ±0.05) (F=498.16,P ＜ 0.01),and the expression levels of protein were separately (1.00 ± 0.14),(0.97 ± 0.11),(0.79 ±0.17),(0.57 ±0.15) (F=390.11,P＜0.01) when the dose of Cr(Ⅵ) at0,0.3,1.2 and 5.0 μmol/L.However,there were no significant changes of expression found in DNMT3b and MBD2.Conclusion Poly-ADP-ribosylation could regulate the activity of DNMT3b and MBD2,protect cells against the DNA methylation alteration induced by Cr(Ⅵ) and maintain the global genomic DNA methylation level.%目的 研究聚-ADP-核糖基化与DNA甲基化在六价铬[Cr(Ⅵ)]致癌过程中的作用,并对两者之间的联系进行探讨.方法 选用前期建立的聚-ADP-核糖水解酶(PARG)缺陷的人支气管上皮细胞(16HBE细胞)作为研究对象,使用Cr(Ⅵ)分别对正常16HBE细胞和PARG缺陷细胞进行处理,通过甲基化免疫荧光技术分析两种不同细胞基因组DNA整体甲基化水平变化,同时检测整体甲基转移酶活性的变化,并进一步利用RT-PCR和western-blotting分别从mRNA和蛋白水平上分析DNA甲基转移酶(DNMT1、DNMT3a、DNMT3b、MBD2)的表达变化.结果 正常16HBE细胞Cr(Ⅵ)处理24h后,基因组DNA甲基化水平降低,且具有剂量-效应关系,而PARG缺陷细胞此种表现不明显；当Cr(Ⅵ)的作用剂量为5.0 μmol/L时,16HBE细胞和PARG缺陷细胞内总体DNA甲基转移酶活性分别为49.33±2.65、80.05±2.05,较对照组(分别为99.27±1.10、99.30±0.60)下降了50％和20％.正常16HBE细胞Cr(Ⅵ)处理24 h后,DNMT1、DNMT3a、DNMT3b、MBD2的mRNA和蛋白水平表达均呈下降趋势.PARG缺陷细胞中DNMT1和DNMT3a的表达出现下调,0、0.3、1.2、5.0 μmol/LCr(Ⅵ)处理组胞内DNMT1 mRNA的相对表达量分别为0.99 ±0.04、0.79±0.05、0.65±0.08、0.51±0.10(F =544.13,P＜0.01),蛋白相对表达量分别为1.00±0.03、0.69±0.15、0.65 ±0.10、0.55±0.13(F=214.12,P＜0.01)；DNMT3a mRNA的相对表达量分别为1.00±0.04、0.93±0.11、0.79±0.07、0.59±0.05(F =498.16,P＜0.01),蛋白相对表达量分别为1.00 ±0.14、0.97 ±0.11、0.79±0.17、0.57 ±0.15(F =390.11,P＜0.01)；但DNMT3b和MBD2表达改变不明显.结论 聚-ADP-核糖基化可通过调节DNMT3b和MBD2的活性,对抗Cr(Ⅵ)诱导的广泛基因组低甲基化,维持细胞基因组整体甲基化水平的稳定.