5-脱氧氮杂胞苷逆转食管鳞癌细胞系p16基因转录表达研究

Reverting transcription of p16 gene in five esophageal squamous cell carcinoma lines by 5-Aza-CdR

目的:探讨5种食管鳞癌细胞株中p16基因转录失活以及去甲基化药物对其作用的机制。方法:采用细胞培养、PCR、DHPLC、MSP、Northern印迹、MTT等方法,检测EC1,EC18,EC109,TE1,TE10食管鳞癌细胞株中p16基因的缺失、突变、甲基化状态和p16的转录表达,观察去甲基化药物5脱-氧氮杂胞苷对p16基因转录表达和细胞增殖能力的影响。结果:除了EC1,其余4种食管鳞癌细胞株均检测出p16基因的不同变化:纯合缺失(EC18),纯合型甲基化(EC18,EC109),杂合型甲基化(TE1,TE10),且纯合缺失的发生率较低(20%),甲基化的发生率较高(80%),经过5-Aza-CdR处理,可见2株发生了杂合型甲基化的TE1和TE10细胞p16基因的转录表达得到了逆转。结论:不同食管鳞癌细胞株p16基因转录失活的原因是不同的,启动子区甲基化为主要原因,其中杂合型甲基化可以被去甲基化药物5-脱氧氮杂胞苷逆转,使p16的转录表达上调。

Objectives：To explore the mechanism of silencing of p16 gene in five esophageal squamous cell carcinoma cell lines;to study the mechanism of reverting transcription of p16 gene in the cell lines by 5-Aza-CdR. Methods： Homozygous deletion, mutation, methylation, mRNA transcription, and protein expression status of p16 in five esophageal squamous cell careinoma lines were examined by cell culture, PCR, DHPLC, MSP, Northern blot, and Western blot, and the effects of 5-Aza-CdR on p16 transcription, expression and proliferation in vitro in the cells were observed. Results： Except EC 1, various changes in pl 6 gene were detected in the other four cell lines： homozygous deletion （ EC 18 ）, biallelic methylation （EC18, EC109）, and monoallelic methylation （TEl, TEl0）. The methylation occurred more frequently（80% ） than homozygous deletion（20% ）. The transcription and expression of p16 were up-regulated in TEl and TE10 cells in which monoallelic methylation occurred. Conclusion： There are different mechanisms of silencing of p16 transcription and expression in different esophageal squamous carcinoma cell lines. The main mechanism of silencing of p16 is methylation of its promoter. 5-Aza-CdR can revert monoallelic methylation and upregulate the transcription and expression of p16 gene.