1,25-二羟维生素D3对子宫内膜癌HEC-1-A细胞增殖与S期激酶相关蛋白及抑癌基因p27表达的影响

目的 观察1,25-二羟维生素D3对人子宫内膜癌HEC-1-A细胞增殖及S期激酶相关蛋白（Skp2）基因、抑癌基因p27表达的影响,探讨其抗癌机制.方法 应用免疫组织化学方法检测人子宫内膜癌HEC-1-A细胞表达维生素D受体（VDR） 选用人子宫内膜癌HEC-1-A细胞进行体外培养,用CCK-8法测定不同浓度1,25-二羟维生素D3作用细胞后不同时间后细胞生长抑制率,流式细胞仪进行细胞周期分析,采用荧光实时定量聚合酶链反应（PCR）及蛋白质印迹法检测Skp2/p27基因表达的变化.结果 人子宫内膜癌HEC-1-A细胞表达VDR,且定位于细胞核内 1×（10-8～10-6）mol/L浓度的1,25-二羟维生素D3作用于HEC-1-A细胞1～5 d范围内各不同浓度处理组细胞生长增殖均受到明显抑制,并且随着作用浓度增加,细胞增殖能力逐渐下降 1,25-二羟维生素D3能增加G0/G1期细胞比例,降低S、G2/M期细胞比例,从而降低细胞增殖指数（P＜0.01） 在mRNA、蛋白质水平上,能降低Skp2表达,而p27mRNA减少,p27蛋白表达明显增加.结论 1,25-二羟维生素D3对人子宫内膜癌HEC-1-A细胞有抑制作用,可能是通过转录水平下调Skp2基因表达,同时由于Skp2的减少增加p27蛋白稳定性,减少其降解,从而产生抑癌作用.

The effect of 1,25-( OH)2D3 on proliferation and expression of Skp2/p27 in endometrial carcinoma cell HEC-1-A

Objective To investigate the expressions of vitamin D receptor (VDR) in endometrial carcinoma cell HEC-1-A;to study the effect of 1,25-(OH)2D3 on prolifetation of human endometrial carcinoma cell HEC-1-A and the mechanism of detecting the expression of S-phase kinase associated protein 2 ( Skp2 ) and p27 gene.Methods Expressions of VDR in human endometrial carcinoma cell HEC-1-A were detected by immunohistochemical method. HEC-1-A human endometrial carcinoma cell line were cultured in vitro and treated with different concentrations of 1,25-( OH)2D3 for 1-5 days. Cell proliferation was evaluated by CCK-8 method, distribution of cell cycle were determined by flow cytometry. The expression level of Skp2/p27 mRNA was examined by real-time quantitative polymerase chain reaction (RT-qPCR). The expression level of Skp2/p27 protein was finally detected by Western blotting. Results (10-8-10-6) mol/L 1,25-(OH)2D3 inhibited the proliferation of HEC-1-A cell line,and inhibitory rate was positively associated with concentration of 1,25-( OH)2D3 and treatment time. 1,25-(OH)2D3 increased the rates of G0/G1, decreased the rates of S and G2/M and also decreased cells PI (P ＜0.01 ). The mRNA and protein expression of Skp2 in HEC-1-A cell lines after 1,25 (OH)2 D3 treatment decreased.The level of p27 mRNA was decreased but the level of p27 protein was significantly increased. Conclusions 1 ,25-(OH)2D3 can inhibit the growth of HEC-1-A cell line. It may inhibit cancer cell proliferation through down-regulation of Skp2 gene and increase p27 stability by decreasing the abundance of Skp2.