3种结肠癌细胞株中DLC-1基因的表达及其启动子区的甲基化状态研究-东南大学学报医学版

3种结肠癌细胞株中DLC-1基因的表达及其启动子区的甲基化状态研究

单位：东南大学基础医学院,病理学与病理生理学系,江苏,南京,210009

分类号：Q754

出版年·卷·期（页码）：2008·27·第一期（6-10）

摘要：

目的:研究候选抑癌基因DLC-1在结肠癌细胞株中的表达及其启动子区的甲基化状态,初步探讨结肠癌细胞株DLC-1基因表达水平与其启动子区甲基化的相关性.方法:采用RT-PCR技术分别检测DLC-1基因在人结肠癌细胞株CaCo-2、HT-29和LoVo中的表达水平;应用甲基化特异性PCR(MSP)方法和亚硫酸氢盐修饰DNA测序检测启动子区域甲基化状态;分别用0、5、10 μmol·L-1浓度的去甲基化药物5氮杂胞苷处理DLC-1基因启动子区域高甲基化状态细胞后,再检测DLC-1基因mRNA表达水平.结果:人结肠癌细胞株CaCo-2和LoVo中DLC-1 mRNA呈阳性表达,HT-29中的表达呈阴性表达;CaCo-2和LoVo细胞DLC-1基因启动子区域未发生甲基化,而HT-29出现启动子区高甲基化状态;经5氮杂胞苷药物干预后,HT-29结肠癌细胞的DLC-1基因表达明显增强.结论:结肠癌细胞株HT-29中DLC-1基因表达沉默与启动子区高甲基化状态有关.

Objective To determine whether aberrant methylation is a contribution factor to transcriptional inactivation of DLC-1 in colon cell lines.Via the investigation of the expression and methylation status of DLC-1(deleted in liver cancer,DLC-1)gene in human colon cell lineswere investigated.Methods Reverse transcipatase PCR was used to explore mRNA expression of the DLC-1 gene.Methylation specific polymerase chain reaction(MSP)and sodium bisulfite genomic sequencing(BSP)were used to test promoter methylation of DLC-1 gene in CaCo-2,LoVo and HT-29.The hypermethylation status was reversed by adding various concentration(0,5,10 μmol·L-1)of 5-aza-2’-deoxycytidine(5-Aza-CdR),a demethylating agent.Results DLC-1 gene was obviously expressed in CaCo-2 and LoVo cell lines,both cell lines showed no methylation.On the contrary,loss of DLC-1 mRNA expression was detected in HT-29 due to the methylation of CpG island of DLC-1 gene.After the treatment of 5-Aza-CdR in hypermethylation cell line HT-29,the expression of DLC-1 mRNA was increased obviously.Conclusion This promoter hypermethylation is correlated with DLC-1 gene expression in human colon cancer HT-29 cell line and plays a key role in DLC-1 silencing.

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