Wip1基因在人脑胶质瘤细胞恶性增殖中的作用及其机制

The roles of Wip1 in malignant proliferation of human brain glioma cells and its mechanisms

目的研究Wip1基因在人脑胶质瘤细胞增殖的作用及机制。方法构建人Wip1基因RNA干扰(RNAi)慢病毒载体,有效沉默胶质瘤细胞U251及U87-MG细胞的Wip1基因表达,检测细胞增殖能力。流式细胞术PI单染法检测Wip1基因沉默后细胞周期分布。实时定量PCR法及Western blotting分别检测差异性基因m RNA及蛋白表达。结果胶质瘤细胞Wip1基因表达被有效沉默。Wip1基因沉默的胶质瘤细胞增殖变慢,在U87-MG细胞更加明显。细胞周期分析显示:Wip1基因沉默对U251细胞周期无明显影响,而U87-MG细胞则在10 d时出现明显S期增多和G1、G2期细胞减少。Wip1基因沉默后,U251细胞中CDKN2A和p14ARF基因表达下调,而在U87-MG细胞中表达均上调。Western blotting结果显示:在U251与U87-MG胶质瘤细胞中p38MAPK表达均上调。在U87-MG细胞中p53及p-p53(Ser 15)表达均上调,但在U251细胞中无明显变化。结论 Wip1对胶质瘤细胞增殖具有重要作用。在U87-MG细胞增殖作用主要与其调节p53功能有关。

Objective To research the roles that Wip1 plays in the malignant growth of glioma cells and explore the possible mechanisms. Methods Glioma cells U251 and U87-MG were infected with Wip1 RNAi lentiviral vector. The proliferation activities of cells with Wip1 silencing were detected. Cell cycle distributions were analyzed by flow cytometry combined with PI staining. The m RNA expression of differentially expressed genes after Wip1 silencing was identified by real-time quantitative PCR, and the expression of protein was identified by Western blotting. Results The expressions of Wip1 gene in glioma cells were silenced effectively. Cells with Wip1 silencing had reduced proliferation ability, especially in U87-MG cells. No significant changes of cell cycle distributions analyzed by flow cytometry were detected in U251 cells after Wip1 silencing. But increased S phase and decreased G1 and G2phases were detected in U87-MG 10 d after the infection. After Wip1 silencing, CDKN2 A and p14 ARF gene m RNA expressions were down-regulated in U251 cells, but there were increased tendencies in U87-MG cells. Western blotting showed that protein expressions of p38 MAPK in both U251 and U87-MG cells were increased. The expressions of both p53 and p-p53（Ser 15） were up-regulated in U87-MG cells but remained unchanged in U251 cells. Conclusions Wip1 plays an important role in proliferation of glioma cells. The role is of much relevance with the fact that Wip1 could inhibit the function of p53 in U87-MG cells.