PTEN基因对子宫内膜癌细胞PI3K/AKT通路的影响

 【目的】探讨PTEN基因在子宫内膜癌PI3K/AKT通路中的作用，评估PTEN基因在抗肿瘤治疗中的意义。【方法】利用慢病毒载体系统, 构建人PTEN基因RNA干扰慢病毒载体和PTEN基因过表达慢病毒载体,分别转染PTEN野生型的HEC-1A细胞和PTEN突变型的Ishikawa细胞, 建立PTEN 基因敲减及过表达的细胞模型，Western Blot方法检测PTEN基因敲减或过表达前后PTEN蛋白的表达和AKT通路的活化情况, MTT法检测慢病毒转染前后细胞增殖的变化,流式细胞术检测慢病毒转染前后细胞周期及细胞凋亡率的变化。【结果】1. HEC-1A细胞转染PTEN基因干扰慢病毒载体后，细胞的增殖曲线和细胞凋亡率与对照组比较无明显变化。但Ishikawa细胞转染PTEN基因过表达慢病毒载体后，增殖曲线和细胞凋亡率显示其生长呈明显抑制状态。2.当HEC-1A细胞转染了RNA干扰病毒后（HEC-1A-RNAi），Western Blot 结果显示PTEN蛋白表达下调，其EGFR, p-EGFR, mTOR, p-mTOR, AKT, p-AKT蛋白表达增强，当Ishikawa细胞转染了过表达载体病毒后（Ishikawa-PTEN），Western Blot 结果显示其PTEN蛋白表达上调，p-EGFR，p-mTOR，AKT，p-AKT蛋白表达减弱。3.HEC-1A-RNAi细胞周期中G1期细胞比例减少，S期细胞比例增加，Ishikawa-PTEN细胞周期中G1期细胞比例增加，S期细胞比例减少。【结论】PTEN 基因可以抑制细胞增殖过程, 有可能成为子宫内膜癌患者基因治疗的选择之一。

Effect of Different PTEN Status on PI3K/AKT Pathway of Endometrial Cancer Cell Lines

【Objectives】 To explore the function of PTEN gene in the PI3K/AKT pathway, to evaluate the function of PTEN gene in the antitumor therapy. 【Methods】 Two lentiviral vectors were constructed: one was used to conduct the RNA interference against PTEN gene, another was used to over-express the wild type PTEN gene. These two vectors were transducted into the two endometrial cancer cell lines with different PTEN status. Cell viability was evaluated by MTT assay. Cell apoptosis and cell cycle were evaluated by flow cytometry. Western blotting was performed to evaluate the related protein alteration in PI3K/AKT signaling pathway after the cell transfection. 【Results】1.When PTEN was knocked down with siRNA on HEC-1A cells (PTEN wild type), there was no significant change in both cell viability and cell apoptosis rate compared with HEC-1A cells. On Ishikawa cells (PTEN mutated), when PTEN gene was over-expressed, the results of cell viability and cell apoptosis rate showed cell proliferation was inhibited obviously; 2.Western blot results demonstrated that PTEN expression in HEC-1A-RNAi cells was abated, expression of EGFR, p-EGFR, mTOR, p-mTOR, AKT, p-AKT were up-regulated, PI3K/AKT pathway was activated. In Ishikawa-PTEN cells with PTEN over-expressed, the results manifested that expression of EGFR, p-EGFR, mTOR, p-mTOR, AKT, p-AKT were down-regulated, PI3K/AKT pathway was inhibited. 3.When PTEN was knocked down, the proportion of cells in G1 phase decreased and the proportion of cells in S phase increased. By contrast, when PTEN over-expressed, the proportion of cells in G1 phase increased and the proportion of cells in S phase decreased.【Conclusions】 PTEN genes can restrain cell proliferation process, might become one of the choices in endometrial carcinoma gene therapy.