慢病毒介导的RNA干扰沉默Wip1基因对人脑胶质母细胞瘤细胞生长的影响

Inhibitory effects of lentivirus mediated RNA interference targeting human Wip1 gene on the proliferation of human glioma U251 cells

目的构建人Wip1基因的RNA干扰（RNAi）慢病毒载体，有效沉默人胶质瘤U251细胞的Wip1基因并观察其对细胞生长的影响。方法设计并合成3条Wip1基因特异性RNAi靶序列，构建到慢病毒pFU—GW—iRNA载体中。慢病毒包装转染HEK293T细胞，获得病毒上清并测定其滴度；感染U251细胞，实时定量聚合酶链反应（PCR）及Westernblot鉴定RNA干扰效率；筛选出基因沉默效率最高的慢病毒感染U251细胞，CCK-8法检测细胞的增殖，Westernblot检测RNA干扰后的bcl-2蛋白表达。结果PCR扩增和测序表明成功构建Wip1慢病毒干扰载体，病毒载体包装获得的病毒上清滴度在3×10^8～8×10^8TU／ml。可以有效地沉默U251细胞中Wip1基因的表达，构建的RNAi慢病毒载体感染U251细胞后Wip1基因的mRNA表达量同对照组比较分别为36．3％、32．9％、23．8％。稳定Wip1RNA干扰后的U251细胞4d后细胞增殖能力下降35．1％。结论慢病毒介导的RNA干扰可以高效稳定地沉默基因表达，Wip1基因促进了U251细胞的增殖。

Objective To construct the lentiviral expression vector for RNA interference （RNAi） of Wip1 gene in glioma cells and provide research foundation for studying the mechanisms that wild-type p53-induced phosphatase 1 （ Wip1 ） gene functions for malignant proliferation of gliomas. Methods Three effective sequences of RNAi targeting Wip1 were confirmed. Both sense and antisense Oligo DNA of the targeting sequence were designed, synthesized and cloned into the pFU-GW-iRNA vector. After cotransfectsd with lentiviral vector of T293 cells,the lentivirus was produced and the titer of virus was tested. U251 cells were infected with the lentivirus and the expression of Wip1 mRNA and protein in U251 cells was detected by real-time quantitative polymerase chain reaction （PCR） and Western blotting. The propagation of U251 was detected by CCK-8 and expression of bcl-2 was detected by Western blotting. Results A recombinant lentiviral vector expressing shRNAs against Wip1 gene was obtained and confirmed by DNA sequencing. The titer of virus was 3 ×10^8-8×10^8 TU/ml. Wip1 mRNA and protein expression in U251 cells after infected with lentiviral vector was decreased remarkably. The mRNA expression was 36. 3% ,32.9% and 23.8% respectively, compared with the control group. And the lentiviral shRNA expression vector reduced cell proliferation by 35. 1% 4 d after stable Wip1 gene knock-down in U251 cells. Conclusion The lentiviral shRNA expression vector targeting human Wip1 gene capable of stable Wip1 gene knock-down in U251 cells has been successfully constructed, which provides a basis for further study of mechanisms that Wip1 gene acts for malignant proliferation of gliomas. And preliminary conclution was drawn that Wip1 gene may promote the growth of U251 ceils.