Numb基因上调对人肾细胞癌细胞周期和增殖的影响

Effect of Numb overexpression on cell cycle and proliferation in human renal cell carcinoma cells

目的 研究Numb基因上调对人肾细胞癌的细胞周期和增殖能力的影响及相关机制.方法 选取人肾细胞癌细胞786-O为研究对象,使用Numb-ORF表达质粒转染细胞为实验组,设置阴性对照及空白对照组.采用荧光定量聚合酶链式反应和蛋白质印迹技术检测各组Numb基因和肿瘤增殖抗原Ki-67的表达;采用流式细胞技术检测细胞周期,计算G0/G1期细胞比例、增殖指数（PI）和S期分数（SPF）;以MTS法检测细胞的增殖活力.结果 实验组Numb的相对表达水平为（18.97±1.49）,显著的高于阴性对照组（3.34±0.41）和空白对照组（3.21±0.39）;实验组Ki-67的相对表达水平为（4.31 ±0.58）,明显少于阴性对照组（10.35±0.84）和空白对照组（9.89±0.73）,差异有统计学意义（P＜0.01）.细胞周期检测：实验组中G0/G1期细胞的比例（％）为60.47±1.58,明显高于阴性对照组（49.67±1.97）和空白对照组（52.52±2.47）（P＜0.01）.实验组的PI和SPF均低于两对照组（P＜0.01）.增殖检测：实验组在24、48、72h的吸光度（A）值均低于两对照组（P＜0.01）.结论 在786-O细胞中上调Numb基因可以抑制Ki-67的表达,减低细胞的PI和SPF,促使细胞发生G0/G1期阻滞,抑制细胞的增殖活力,实验结果提示Numb基因在肾细胞癌中可能发挥抑癌因子作用.

Objectives To investigate the effect of overexpressed Numb on cell cycle and proliferation in human renal cell carcinoma cells and its related mechanism.Methods The Numb-ORF plasmids were transfected into renal cell carcinoma cells 786-O, set the negative control and blank control.The expression of Numb and Ki-67 was detected by Real-Time PCR and Western Blot.Then cell cycle and proliferation were analyzed by Flow cytometry and MTS assay respectively.Results Compared to the negative control group （3.34 ± 0.41） and blank control group （3.21± 0.39）,Numb expression in the Numb-ORF group （18.97 ± 1.49） was significantly higher （P 〈 0.01）.Meanwhile, Ki-67 level was decreased ： the Numb-ORF group was（4.31 ± 0.58）, negative control was（10.35-± 0.84）, blank control was （9.89-± 0.73, P 〈 0.01）.Flow cytometry analysis showed that： in the Numb-ORF group, G0/G1 cells（%） was（60.47 ± 1.58）, significantly higher than the negative control group（49.67 ± 1.97） and blank control group（52.52 ±2.47, P 〈0.01）.And Proliferation Index （PI） and S Phase Fraction （SPF） in the Numb-ORF group were lower than the other two controls（P 〈0.01）.In MTT assay, the absorption value of Numb-ORF group was significantly reduced at the time points of 24,48 and 72h （P 〈 0.01）.Conclusions Overexpression of Numb could downregulate Ki-67 expression, reduce PI and SPF, induce G0/G1 phase arrest, and inhibit proliferation of 786-O cells.Moreover, Numb may function as a suppressor in human renal cell carcinoma.