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  

Promoter methylation and expression of DLC-1 gene in human colon cell lines

作　　者：金月玲[1] 田小强[1] 商延芳[1] 黄培林[1] 

机构地区：[1]东南大学基础医学院病理学与病理生理学系,江苏南京210009

出　　处：《东南大学学报（医学版）》2008年第1期6-10,共5页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(30471937)

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、10μmol.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.

关 键 词：DLC-1基因 甲基化 结肠癌细胞株 甲基化特异性PCR 

分 类 号：Q754[生物学—分子生物学]