摘要
目的探讨Wnt抑制性基因甲基化对结直肠癌细胞株Wnt/β-catenin信号通路的影响。方法使用甲基特异性PCR和逆转录实时定量PCR方法,分别检测结直肠癌细胞株COLO 320、HT-29及正常细胞株CCD-18Co中WIF-1、DKK-1、DKK-3的启动子Cp G岛甲基化、mRNA水平,和测定β-catenin蛋白磷酸化情况,并观察5-氮杂-2'-脱氧胞苷(5-aza-d C)去甲基化对各指标的影响。结果 COLO 320细胞的DKK-1及DKK-3 mRNA水平明显降低、甲基化程度高;HT-29细胞的DKK-3、WIF-1 mRNA水平低、甲基化程度高;两个肿瘤细胞株的β-catenin蛋白总量均明显增高,且主要为非磷酸化的状态。5-aza-d C可减少这些指标的改变。结论抑制性基因甲基化调节的Wnt/β-catenin通路的异常激活,可能在结直肠癌的形成中有重要作用。在不同的肿瘤细胞株之间、不同Wnt抑制基因的甲基化程度存在差异;该领域的深入研究有助于开发出新的治疗药物。
Objective To reveal the the effect of methylated inhibitory genes on Wnt / β-catenin pathway in colorectal adenocarcinoma cell lines. Methods With colorectal adenocarcinoma cell lines COLO 320,HT-29 and normal colorectal cell line c CCD-18 Co,methylation status of Cp G island in promotor of WIF-1,DKK-1 and DKK-3 genes were determined by methylation specific PCR（ MSP）,and mRNA levels of these genes were quantified by real-time RT-PCR,and phosphorylated β-catenin were semi-quantified by Western blot. Effect of DNA-demethylating agent 5-aza-2＇-deoxycytidine（ 5-aza-d C） treatment were also evaluated. Results DKK-1and DKK-3 mRNA decreased accompanied with hypermethylation status of the Cp G islands in COLO 320,while DKK-3 and WIF-1 mRNA decreased accompanied with hypermethylation in HT-29. Total β-catenin increased significantly while phosphorylated β-catenin declined in both cell lines. 5-aza-d C ameliorated these aberrant parameters partially. Conclusion The aberrant activation of Wnt / β-catenin pathway mediated by the hypermethylation of WIF-1 and DKKs genes may contribute to the oncogenesis of colorectal adenocarcinoma,while the methylation level of different Wnt inhibitory genes varied in colorectal cancer cell lines. Further research on this field will prompt the development of new therapies targeting the methylation process.
出处
《中南医学科学杂志》
CAS
2015年第4期383-387,共5页
Medical Science Journal of Central South China
基金
衡阳市科学技术局科技计划(编号:2011KJ64)
