人食管鳞癌细胞株中RIZ1基因启动子区甲基化状态的研究

Study on RIZ1 gene promoter methylation status in esophageal carcinoma cell lines

目的:探讨6种人食管鳞癌细胞株KYSE150,KYSE510,TE13,EC9706,CaEs17和EC109中RIZ1基因启动子区的甲基化状态;观察特异性DNA甲基转移酶抑制剂5-Aza-CdR对存在甲基化的细胞株RIZ1基因的转录调控作用及其对该细胞生长增殖的影响。方法:甲基化特异PCR(MSP)法检测6种人食管鳞癌细胞株中RIZ1基因启动子区的甲基化状态;实时定量PCR法检测5-Aza-CdR处理前后RIZ1基因启动子区CpG岛高度甲基化的人食管鳞癌细胞株TE13中RIZ1 mRNA表达量的变化;MTT法检测5-Aza-CdR对TE13细胞生长增殖的影响。结果:(1)6种人食管鳞癌细胞株中TE13,CaEs17,EC109三株细胞RIZ1基因启动子区存在甲基化;(2)5-Aza-CdR处理细胞株TE13后,RIZ1 mRNA表达量上调;(3)5-Aza-CdR能抑制TE13的生长增殖,并随时间的延长和浓度的增加抑制作用变明显。结论:启动子区CpG岛高度甲基化是人食管鳞癌细胞株TE13 RIZ1基因表达沉默的重要原因;5-Aza-CdR能使TE13 RIZ1 mRNA表达上调;并能抑制TE13细胞的生长增殖,并呈时间和浓度依赖性。

Objective： To investigate the promoter region methylation status of RIZ1 gene in the human esophageal squamous carcinoma cell lines of KYSE150, KYSE510, TEl3, EC9706, CaEsl7 and EC109, and the influence of methyltransferase inhibitor 5-Aza-CdR on the transcription of RIZ1 gene in the cell line whose RIZ1 gene promoter region hypermethylation, and its influence on the cell prolifera- tion. Methods： （ 1 ）Methylation-specific PCR（MSP） was used to investigate the promoter region methylation status of RIZ1 gene in the six human esophageal squamous cell carcinoma cell lines, and chose one cell line whose RIZ1 gene promoter region hypermethylation was detected for the next studies. （2）5-Aza-Cdr was treated to the cell line TEl3 whose RIZ1 gene promoter region hypermethylation was detected. Real-time PCR was used to investigate its influence on the transcription of RIZ 1 gene. （3）M＇IT was used to detect if 5-Aza-CdR inhibit the proliferation of the cell line whose RIZ1 gene promoter region hypermethylation was detected. Results： （ 1 ）Promoter hypermath- ylation of RIZ1 gene was detected in TE13,CaEsl7,EC109, chose the cell line TEl3 for the next studies. （2）The expression of RIZ1 mRNA in TE 13 was up-regulated after treated by 5-Aza-CdR. （3）5-Aza-CdR inhibited the cell proliferation of TE 13 in a time and concentration-dependent manner. Conclusion： Promoter hypermethylation may play an important role in the epigenetic silencing of RIZ1 gene expression. 5-Aza-CdR could up-regulate the expression of the RIZ1 mRNA in the cell line TEl3, and it could inhibit the cell pro- liferation of TE 13.