论文部分内容阅读
目的通过检测氯乙烯(VC)染毒大鼠肝细胞RASSF1A基因启动子区甲基化的水平及mRNA的表达量,探讨RASSF1A基因在氯乙烯致癌过程中的表观遗传机制。方法选取96只健康雄性SD大鼠,随机分为4组:阴性对照组(0mg/kg)和低剂量(5 mg/kg)、中剂量(25 mg/kg)、高剂量(125 mg/kg)3个氯乙烯染毒组。腹腔注射,隔日染毒,每周3次。每组分别于6、8和12周随机处死8只,取其肝脏。采用甲基化特异性实时荧光定量(QMSP)检测大鼠肝细胞RASSF1A基因启动子区甲基化的水平,采取实时荧光定量(QPCR)测定RASSF1A mRNA表达量的改变。结果染毒6周时,RASSF1A基因启动子区甲基化水平各组间无明显变化,差异无统计学意义(P>0.05);但RASSF1A mRNA的表达量随着剂量的增加而升高,且高剂量组、中剂量组与对照组相比,差异有统计学意义(P<0.05)。染毒8周时,RASSF1A基因启动子区甲基化水平和mRNA的表达量在各组间差异无统计学意义(P>0.05)。染毒12周时,RASSF1A基因启动子区甲基化水平随着染毒剂量的增加而明显升高,高剂量组与其他各组相比,差异有统计学意义(P<0.05);mRNA的表达量随着染毒剂量的增加而出现下降,且对照组和低剂量组与高剂量组相比,差异有统计学意义(P<0.05)。此外,在高剂量组中,8周时RASSF1A启动子区甲基化水平较6周时低,差异有统计学意义(P<0.05),而12周时启动子区甲基化水平较6周时高,且差异有统计学意义(P<0.05);mRNA表达量随着染毒时间的延长而下降,且8和12周mRNA表达量与6周时相比,差异有统计学意义(P<0.05),在其余各组中,不同染毒时间下RASSF1A甲基化水平及mRNA表达量均未出现统计学差异(P>0.05)。结论氯乙烯长期染毒可致大鼠肝细胞RASSF1A基因启动子区甲基化水平的升高,并引起其mRNA的表达量下降,可能参与氯乙烯致癌的表观遗传机制。
Objective To investigate the epigenetic mechanism of RASSF1A gene in the carcinogenesis of vinyl chloride by detecting the methylation level of RASSF1A promoter and the mRNA expression of RASSF1A gene in the rat VC cells exposed to vinyl chloride (VC). Methods Ninety-six healthy male Sprague-Dawley rats were randomly divided into 4 groups: negative control (0 mg / kg) and low dose (5 mg / kg) Three vinyl chloride exposure groups. Intraperitoneal injection, the next day exposure to poison, 3 times a week. Eight rabbits were randomly sacrificed at 6, 8 and 12 weeks respectively, and their livers were harvested. The methylation level of RASSF1A gene promoter region in rat hepatocytes was detected by methylation-specific real-time fluorescence quantitative (QMSP), and the changes of RASSF1A mRNA expression were detected by real-time fluorescence quantitative PCR (QPCR). Results There was no significant difference in the methylation level of RASSF1A promoter between the groups at 6 weeks after exposure (P> 0.05). However, the expression of RASSF1A mRNA increased with increasing dose Compared with the control group, the difference was statistically significant (P <0.05). At 8 weeks of exposure, there was no significant difference in methylation level and mRNA expression of RASSF1A promoter among all groups (P> 0.05). At 12 weeks of exposure, the methylation level of RASSF1A promoter region was significantly increased with the increase of exposure dose, and there was significant difference between high dose group and other groups (P <0.05). The mRNA level of mRNA The expression level decreased with the increase of exposure dose, and there was significant difference between the control group and the low dose group and the high dose group (P <0.05). In addition, the methylation level of RASSF1A promoter in the high-dose group was lower at 8 weeks than that at 6 weeks (P <0.05), but at 12 weeks, the level of methylation in the promoter region was 6 weeks (P <0.05). The mRNA expression decreased with the prolongation of exposure time, and there was significant difference between 8 and 12 weeks mRNA expression (P <0.05). In the other groups, there was no significant difference in RASSF1A methylation and mRNA expression between different exposure groups (P> 0.05). Conclusion Long-term exposure to vinyl chloride can cause the increase of methylation level of RASSF1A promoter in rat hepatocytes and decrease the mRNA expression of RASSF1A, which may be involved in the epigenetic mechanism of carcinogenesis of vinyl chloride.