转染细胞周期蛋白E1对人脑神经胶质瘤细胞增殖和迁移能力的影响

Effect of Cyclin E1 on Proliferation and Migration of Human Glioma Cells

目的探讨人脑神经胶质瘤U251的细胞活性与细胞周期蛋白E1(Cyclin E1)表达的关系.方法pEGFPTimp1-Cyclin E1质粒体外转染上调U251细胞中Cyclin E1表达为Cyclin E1转染组,pEGFP-Timp1空载体质粒转染U251细胞为阴性对照组,常规培养U251细胞为空白对照组,荧光显微镜下观察并鉴定.CCK-8法检测细胞增殖能力;Transwell实验检测细胞迁移能力;RT-PCR检测Cyclin E1 mRNA表达水平;Western blot检测Cyclin E1、p-PI3K、p-Akt蛋白表达水平.结果Cyclin E1转染组Cyclin E1 mRNA和蛋白表达水平明显高于阴性对照组和空白对照组,差异具有统计学意义(P<0.01).U251细胞培养12、24 h时增殖水平在3组间比较差异无统计学意义(P>0.05);培养48、72、96 h时,与阴性对照组和空白对照组比较,U251细胞增殖水平明显升高,差异具有统计学意义(P<0.05).Cyclin E1转染组的迁移细胞数为(528.05±51.83)个,明显多于阴性对照组(131.73±35.09)个、空白对照组(129.58±32.14)个,差异具有统计学意义(P<0.01).Cyclin E1转染组p-PI3K、p-Akt蛋白表达水平明显高于阴性对照组和空白对照组,差异具有统计学意义(P<0.01);阴性对照组与空白对照组p-PI3K、p-Akt蛋白表达水平比较差异无统计学意义(P>0.05).结论Cyclin E1表达上调能够明显增强人脑神经胶质瘤U251细胞的增殖和迁移能力,可能与激活PI3K/Akt信号通路有关.

Objective To investigate the relationship between the activity of U251 cells and the expression of Cyclin E1.Method U251 cells were transfected with pEGFP-Timp1-Cyclin E1 plasmid in vitro to upregulate Cyclin E1 expression,U251 cells were transfected with pEGFP-Timp1 empty vector plasmid as negative control group,and U251 cells were cultured as blank control group.CCK-8 method was used to detect cell proliferation;Transwell test was used to detect cell migration;RT-PCR was used to detect Cyclin E1 mRNA expression;Western blot was used to detect Cyclin E1,p-PI3k and p-Akt protein expression.Results The expression level of Cyclin E1 mRNA and protein in the Cyclin E1 transfection group was significantly higher than that in the negative control group and the blank control group(P<0.01).When U251 cells were cultured for 12 hours and 24 hours,there was no significant difference among the three groups(P>0.05);When they were cultured for 48 hours,72 hours and 96 hours,the proliferation level of U251 cells was significantly higher than that of the negative control group and the blank control group(P<0.05).The number of migrating cells in Cyclin E1 transfection group was 528.05±51.83,which was significantly higher than that in negative control group(131.73±35.09)and blank control group(129.58±32.14)(P<0.01).The expression level of p-PI3k and p-Akt protein in Cyclin E1 group was significantly higher than that in negative control group and blank control group(P<0.01),but there was no significant difference between negative control group and blank control group(P>0.05).Conclusion Up-regulation of Cyclin E1 expression can significantly enhance the proliferation and migration of human glioma U251 cells,which may be related to activation of PI3K/Akt signaling pathway.