5-Aza-dC和TSA对人喉癌细胞系中CHFR基因启动子甲基化及mRNA表达水平的影响

Effects of 5-Aza-2′-deoxycitydine and trichostatin A on expression and methylation of CHFR in human laryngreal carcinoma cell line

目的:探讨5-杂氮-2′-脱氧胞苷(5-Aza-dC)和制霉菌素A(TSA)对人喉癌细胞系中CHFR基因启动子区甲基化与mRNA表达水平的影响。方法:采用荧光定量PCR技术和甲基化特异性PCR技术检测5-Aza-dC和TSA处理前后的Hep-2人喉癌细胞系基因CHFR启动子区甲基化与mRNA表达水平的变化。结果:在对照组Hep-2人喉癌细胞系中CHFR基因启动子区显示DNA甲基化,用5-Aza-dC作用后DNA甲基化程度明显减弱,mRNA表达量上调(1.75±0.21);用TSA作用后DNA甲基化程度和mRNA表达量(1.05±0.13)无明显变化。联合使用5-Aza-dC和TSA后DNA甲基化程度明显减弱,mRNA表达量明显上调(2.15±0.18)。结论:CHFR基因启动子区DNA甲基化在喉癌的发生、发展中是一个频发事件,是导致其mRNA表达下调的主要原因,5-Aza-dC单独应用和联合TSA能够逆转DNA甲基化状态,从而调控其基因表达,为临床使用药物治疗喉癌提供了新思路。

Objective：To explore the effects of 5-Aza-2′-deoxycitydine（5-Aza-dC） and trichostatin A（TSA） on the expression and methylation of CHFR in human laryngreal carcinoma cell line.Method：The mRNA expression and promoter hypermethylation and were detected by Realtime fluro-genetic quantitative PCR and methylation specific PCR in Hep-2 cell line,which were cultured in vitro and then treated with different concentrations of 5-Aza-dC and TSA.Result：Compared with the control team,5-Aza-dC alone reactivated expression of the CHFR in Hep-2 cell line（1.75±0.21）.TSA had no effect on gene expression（1.05±0.13）.The combined treatment with 5-Aza-dC and TSA increased gene expression（2.15±0.18）.The cell lines showed a characteristic DNA methylation status.5-Aza-dC and combined 5-Aza-dC and TSA resulted in demethylation of CHFR.In contrast,TSA alone did not affect the DNA methylation status of CHFR.Conclusion：Hypermethylation of CHFR gene promoter is a common event in the occurrence and development of laryngreal carcinoma.The promoter aberrant methylation of CHFR is a main cause for down-expression of CHFR.After either treatment with 5-Aza-dC alone or in combination with TSA,the expression of CHFR is up-regulated duo to the reversal methylation.It can be a new idea to the therapy of laryngreal carcinoma.