5-氮杂-2’-脱氧胞苷抑制肾癌细胞增殖及逆转γ-catenin基因甲基化作用

Effect of 5-Aza-2’-deoxycytidine on growth inhibition of renal carcinoma cell line OS-RC-2 and reversion of γ-catenin gene hypermethylation

目的:研究甲基化抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-CdR)对人肾癌OS-RC-2细胞生长的抑制作用及对γ-catenin基因甲基化状态的影响,初步探讨肾癌发病机制及临床治疗的可行性。方法:不同浓度5-Aza-CdR处理肾癌细胞OS-RC-2后,采用倒置显微镜观察5-Aza-CdR处理OS-RC-2肾癌细胞株前后细胞形态学变化;四唑蓝(MTT)比色观察细胞经药物处理前后的生长活性;流式细胞仪检测5-Aza-CdR对OS-RC-2肾癌细胞株凋亡的影响;Western blot检测5-Aza-CdR处理OS-RC-2肾癌细胞株后γ-catenin蛋白表达的变化;甲基化特异性PCR(methylation-specific PCR,MSP)检测细胞处理前后γ-catenin基因的甲基化状态。结果:形态学观察显示,用药后癌细胞体积缩小,核固缩,染色质凝聚成块;5-Aza-CdR明显抑制肾癌细胞OS-RC-2的生长;细胞凋亡率增高;药物处理后γ-catenin蛋白表达恢复;未经5-Aza-CdR处理的OS-RC-2细胞中γ-catenin基因启动子区域高甲基化,经10-5mol/L5-Aza-CdR处理72h后,γ-catenin基因启动子区域高甲基化得到逆转。结论:5-Aza-CdR能有效逆转肾癌OS-RC-2细胞γ-catenin基因的异常甲基化,恢复γ-catenin蛋白表达,诱导肾癌细胞凋亡。

Objective：To investigate the effects of 5-Aza-2’-deoxycytidine（5-Aza-CdR） a methylation inhibitor on the growth of human renal carcinoma cell line OS-RC-2,and the DNA CpG island demethylation of γ-catenin gene so as to provide insights into origin of renal carcinoma and the possibility of its application in clinical treatment. Methods：MTT method and flow cytometry were used to detect the growth and appoptosis of OS-RC-2 after 5-Aza-CdR treatment respctirely. The western blot was used to detevt γ-catenin protein levels in the cell line before and after treatment with 5-Aza-CdR.The methylation and demethylation status of γ-catenin gene were volume detected by methylation special PCR（MSP）. Results：The morphological pattern changes were observed invert-microscope. OS-RC-2 cells treated with 5-Aza-CdR displayed a slowed growth in comparision with the control cells. The apoptotic rate was also increased after 5-Aza-CdR treatment. Western blot indicated that expression of γ-catenin protein was recovered by 5-Aza-CdR treatment. The high methylation status of γ-catenin gene promoter region was reversed with 10^-5 mol/L 5-Aza-CdR treated for 72 h. Conclusion：5-Aza-CdR effectively causes the demethylation of γ-catenin gene CpG-rich promoter regions, and recover the γ-catenin protein expression, subequenely induce the appoptosis of OS-RC-2 cells.