靶向CK2α基因的siRNA对结肠癌HCT116细胞生长抑制作用及其机制

摘要

目的：探讨RNA干扰酪蛋白激酶2（CK2α）基因表达后对 HCT116细胞生长的抑制作用并阐明其作用机制。方法：针对CK2α的 mRNA序列设计 CK2α-siRNA序列，将体外培养的 HCT116细胞分为正常对照组（未转染）、阴性对照组（转染siRNA）和CK2α-siRNA组（转染CK2α-siRNA），应用Lipofectamine 2000进行转染，利用 Western blotting 法检测CK2α、cyclin H、P53和P21蛋白表达水平；应用 MTT法检测各组 HCT116细胞增殖；采用流式细胞术检测细胞周期时相的分布。结果：与阴性对照组比较， CK2α-siRNA组 CK2α和细胞周期蛋白 cyclin H的表达水平明显降低（P＜0.01），P53蛋白表达水平无明显变化（P＞0.05），P21蛋白表达水平则明显升高（P＜0.01）；MTT检测，与阴性对照组比较，转染48和72 h，CK2α-siRNA组细胞存活率明显降低（P＜0.01）；流式细胞术分析，与阴性对照组比较，CK2α-siRNA组 S期细胞所占比例逐渐减少（P＜0.01），G1期细胞则明显增加（P＜0.01），细胞滞留在 G1期。结论：RNA干扰CK2α表达能够抑制 HCT116细胞增殖，发生 G1期阻滞；其机制可能与 RNA干扰CK2α表达后 cyclin H表达下调及P53活性恢复有关。%Objective To investigate the inhibitory effect of siRNA targeting casein kinase 2 (CK2α)gene on the growth of HCT1 1 6 cells and to clarify its mechanism.Methods CK2α-siRNA sequence was designed according to mRNA sequence of CK2α. The in vitro cultured HCT1 1 6 cells were divided into normal control group (without transfection),negative control group (transfected with siRNA)and CK2α-siRNA group (transfected with CK2α-siRNA ),and the HCT116 cells were transfected with Lipofectamine 2000.The expression levels of CK2α,cyclin H,P53,and P21 proteins in the HCT116 cells were detected by Western blottting method,and the proliferation activities of the HCT116 cells were detected by MTT method,and the cell cycle was measured by flow cytometry. Results Compared with negative control group,the expression levels of CK2α,and cyclin H proteins in CK2α-siRNA group were decreased(P<0.01);the expression level of P53 protein had no change dramatically(P>0.05), and the expression level of P21 protein was increased significantly(P<0.01).Compared with negative control group,the survival rate in CK2α-siRNA group was decreased markedly 48 and 72 h after transfection detected by MTT method(P<0.01).Flow cytometry analysis showed the percent of the cells at G1 phase in CK2α-siRNA group was significantly higher than that in negative control group and the percent of the cells at S phase in CK2α-siRNA group was lower than that in negative control group(P<0.01),and the cell cycle was arrested at G1 phase. Conclusion siRNA targeting CK2αcan inhibit the proliferation of HCT116 cells and induce the arrest of G1 phase, which may be associated with inhibiting the expression of cyclin H and recovering the P53 activity after silencing CK2α.