5-氮杂-2'-脱氧胞苷抑制肾癌细胞增殖的机制研究  

5-aza-2'-deoxycytidine inhibits proliferation in renal carcinoma cells via suppressing methyltransferases and enhancing P21

作　　者：熊浩君[1] 赵凯[2] 何凤田[2] 曾益军[2] 陈姗[2] 陈丙波[1] 

机构地区：[1]第三军医大学实验动物中心,重庆400038 [2]第三军医大学基础医学部生物化学与分子生物学教研室,重庆400038

出　　处：《第三军医大学学报》2014年第9期847-851,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(81273226);重庆市科技攻关重点项目(CSTC2011ggB10014;CSTC2011AB1045);重庆市自然科学基金(CSTC2009BB5171);教育部留学回国人员科研启动基金(2011)~~

摘　　要：目的探讨5-氮杂-2'-脱氧胞苷(5-Aza-CdR)抑制肾癌细胞增殖的分子机制。方法采用不同浓度的5-Aza-CdR作用于769-P和ACHN 2种肾癌细胞,处理72 h后CCK-8法检测其对细胞增殖的影响;5-Aza-CdR处理769-P细胞48 h后,流式细胞仪测定细胞周期分布,RT-PCR测定甲基化转移酶DNMT1、DNMT3a、DNMT3b以及细胞信号抑制因子3(suppressor of cytokine signaling 3,SOCS3)的表达,Western blot测定SOCS3、P21在蛋白质水平的表达;siRNA干扰SOCS3表达以后,加5-Aza-CdR处理769-P细胞48 h,Western blot检测细胞内SOCS3、P21的表达。结果 5-Aza-CdR可以剂量性依赖地抑制肾癌细胞系769-P和ACHN的增殖(P<0.05),且使769-P细胞阻滞在G2/M期。RT-PCR检测发现,5-Aza-CdR可以下调DNMT1和DNMT3a的表达和上调SOCS3的表达(P<0.05)。Western blot检测发现5-Aza-CdR可以促进SOCS3蛋白质的表达,并且伴随着P21蛋白的升高。在干扰SOCS3的表达后,用5-Aza-CdR处理769-P细胞48 h,P21的表达明显降低。结论 5-Aza-CdR通过抑制甲基化转移酶,增强SOCS3介导P21的表达,从而抑制肾癌细胞769-P增殖。Objective To investigate the underlying molecular mechanism of 5-aza-2′-deoxycytidine （5-Aza-CdR） in inhibition of renal carcinoma cell proliferation. Methods Cell proliferation was assessed by CCK-8 assay after the renal carcinoma cell lines 769-P and ACHN were treated with 5-Aza-CdR at different concentrations for 72 h. Flow cytometry was used to detect cell cycle in 769-P cells after treatment of 5-Aza-CdR at different concentrations for 48 h. RT-PCR was used to detect the expression of methyltransferases DNMT1/3a/3b and suppressor of cytokine signaling 3 （SOCS3）. The expression of P21 and SOCS3 at protein level was detected by Western blot analysis, and detected again in the 769-P cells with SOCS3 knockdown by small interfering RNA （siRNA） after 48 hours’ treatment of 5-Aza-CdR at different concentrations. Results CCK-8 assay revealed that 5-Aza-CdR remarkably inhibited the proliferation in the 2 cell lines in a dose-dependent manner （P〈0.05）. 5-Aza-CdR treatment also resulted in the 769-P cells arrested at G2/M phase. RT-PCR showed 5-Aza-CdR down-regulated DNMT1 and DNMT3a but up-regulated SOCS3 （P〈0.05）. Western blotting found that 5-Aza-CdR enhanced the expression of SOCS3 and P21. After knocking down the expression of SOCS3 of 769-P cells, we treated 769-P cells with 5-Aza-CdR for 48 h and found that the up-regulation of P21 was weakened compared with control group. Conclusion 5-Aza-CdR suppresses DNA methyltransferases, and enhances the expression of P21 mediated by SOCS3, and then inhibits the proliferation in renal carcinoma 769-P cells.

关 键 词：5-氮杂-2'-脱氧胞苷 肾细胞癌 细胞增殖 细胞信号抑制因子3 P21 

分 类 号：R730.23[医药卫生—肿瘤] R737.11[医药卫生—临床医学]