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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 molecular mechanism of 5-Aza-2’-deoxycytidine (5-Aza-CdR) inhibiting the proliferation of renal cell carcinoma. Methods The effects of 5-Aza-CdR on 769-P and ACHN cells were detected by CCK-8 assay after 72 h treatment. Cell proliferation was measured by 5-Aza-CdR h, the cell cycle distribution was determined by flow cytometry. The expressions of DNMT1, DNMT3a, DNMT3b and SOCS3 were detected by RT-PCR. The expressions of SOCS3, P21 At the protein level, 769-P cells were treated with 5-Aza-CdR for 48 h after siRNA interference with SOCS3 expression. Western blot was used to detect the expression of SOCS3 and P21. Results 5-Aza-CdR could inhibit the proliferation of human renal cell carcinoma cell line 769-P and ACHN in a dose-dependent manner (P <0.05), and blocked the G2 / M phase of 769-P cell line. The results of RT-PCR showed that 5-Aza-CdR could down-regulate the expression of DNMT1 and DNMT3a and up-regulate the expression of SOCS3 (P <0.05). Western blot showed that 5-Aza-CdR promoted the expression of SOCS3 protein accompanied by the increase of P21 protein. After interfering with the expression of SOCS3, 769-P cells were treated with 5-Aza-CdR for 48 h, the expression of P21 was significantly decreased. Conclusions 5-Aza-CdR can inhibit the proliferation of renal carcinoma cell line 769-P by inhibiting the methylation of transferase and enhancing the expression of P21 mediated by SOCS3.