靶向沉默Wip1基因表达对胶质瘤细胞增殖及放疗敏感性的作用

Effect of targeted silencing Wip1 gene on glioma cells proliferation and sensitivity to radiotherapy

目的探讨靶向沉默Wip1基因表达对脑胶质瘤细胞增殖及放疗敏感性的影响。方法体外培养人胶质瘤细胞株U251,用携带Wip1基因RNA干扰载体的慢病毒感染U251细胞沉默Wip1基因表达,然后对U251胶质瘤细胞进行放疗干预。根据对U251细胞处理方法分为4组:Wip1基因沉默后放疗组(IR+Wip1组)、单纯Wip1基因沉默组(Wip1组)、单纯放疗组(IR组)和NC组(空载体病毒感染U251细胞)。CCK-8法检测细胞的增殖,实时定量PCR检测细胞Chk1、Chk2 mRNA表达,免疫印迹检测细胞Chk1、Chk2蛋白表达。结果 U251细胞慢病毒感染后3~4 d,采用荧光显微镜观察,根据绿色荧光蛋白阳性表达判定感染效率,结果显示感染效率均90%以上。放疗后24、48、72、96、120 h,IR组和Wip1组增殖率均显著低于NC组(P<0.05),而IR+Wip1组细胞增殖率明显低于IR组和Wip1组(P<0.05)。放疗后24 h,IR+Wip1组Chk1 mRNA表达明显低于Wip1组(P<0.05),明显高于IR组和NC组(P<0.05);IR+Wip1组Chk2 mRNA表达明显低于其他3组(P<0.05)。IR+Wip1组Chk1蛋白表达明显低于IR组和NC组(P<0.05),Chk2蛋白表达低于其他3组(P<0.05)。结论靶向沉默Wip1基因表达可抑制胶质瘤细胞的增殖,并可能通过抑制Chk1、Chk2的蛋白表达增加胶质瘤细胞对放疗的敏感性。

Objective To investigate the effects of targeted silencing of Wip1 gene on glioma cells proliferation and sensitivity toradiotherapy. Methods Glioma U251 cells which were cultured were transfected with Wip1 RNAi lentiviral vector. Then the U251 cellswere treated with ionizing radiation （IR） and divided into negative control （NC） group, IR group, targeted silencing Wip1 gene （Wip1）group and IR＋Wip1 group. The proliferation activities of U251 cells, Chk1 and Chk2 mRNA expressions and Chk1 and Chk2 proteinexpressions were detected respectively by CCK-8 kit, real time PCR and Western-blot in all the groups. Results The proliferation ratesin the Wip1 and the IR groups were significantly lower than that in NC group 24, 48, 72, 96 and 120 hours after the radiotherapy （P〈0.05）. The proliferation rate was significantly lower in the IR＋Wip1 group than those in all the other groups 24, 72, 96 and 120 hoursafter the radiotherapy （P〈0.05）. The level of chk1 mRNA expression was significantly lower in IR＋Wip1 group than that in Wip1 groupand higher than those in IR and NC groups 24 hours after the radiotherapy （P〈0.05）. The level of chk2 mRNA expression wassignificantly lower in IR ＋ Wip1 group than those in the other three groups （P〈0.05）.The level of Chk1 protein expression wassignificantly lower in IR＋Wip1 group than those in IR and NC groups 24 hours after the radiotherapy （P〈0.05）. The level of Chk2 proteinexpression was significantly lower in the IR＋Wip1 group than those in the other three groups （P〈0.05）. Conclusions It is suggested thattargeted silencing Wip1 gene can inhibit the proliferation of glioma cells and may increase the sensitivity of glioma cells to radiotherapyby inhibiting the protein expressions of Chk1 and Chk2.