Numb基因上调对人肾细胞癌细胞周期和增殖的影响

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.

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

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.