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目的:应用RNA干扰(RNAi)方法下调胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因表达,研究GFAP对星形胶质瘤细胞U251细胞增殖的影响及可能的作用机制。方法:采用小分子RNA干扰(Small interfering RNA,si RNA)技术特异性的干扰U251细胞中内源性GFAP的表达,并通过RT-PCR和Western blot技术检测U251细胞中GFAP的m RNA和蛋白表达水平,确定干扰效率。Western blot技术检测si-NC和si-GFAP转染48 h后,U251细胞中GFAP、CREB、cyclin D1、CDK2、CDK4、CDK6、p15、p18、p27、Bax、Bcl-2和Caspase 3蛋白表达变化;MTT检测各组的细胞增殖情况。结果:(1)在U251细胞中特异性的抑制GFAP表达,抑制细胞增殖,降低细胞总RNA和总蛋白含量(si-NC组vs.si-GFAP组:t=3.962,P=0.017;t=3.833,P=0.019)。(2)在U251细胞中干扰GFAP表达,显著性抑制CREB(si-NC组vs.si-GFAP组:t=4.834,P=0.008)、cyclin D1(si-NC组vs.si-GFAP组:t=3.610,P=0.023)、CDK2(si-NC组vs.si-GFAP组:t=6.678,P=0.003)和CDK4(si-NC组vs.si-GFAP组:t=4.530,P=0.011)的表达水平;并且显著性提高细胞周期依赖性激酶抑制因子p18(si-NC组vs.si-GFAP组:t=-3.339,P=0.029)和p27(si-NC组vs.si-GFAP组:t=13.859,P=0.000)的蛋白表达水平。(3)MTT检测结果显示24和48 h时si-GFAP组细胞生长速度明显低于si-NC组(si-NC组vs.si-GFAP组:t=6.389,P=0.000;t=6.058,P=0.000),差异有统计学意义。(4)在U251细胞中抑制GFAP表达,对凋亡相关蛋白Bax(si-NC组vs.si-GFAP组:t=-0.657,P=0.547)、Bcl-2(si-NC组vs.si-GFAP组:t=-0.991,P=0.378)和Caspase 3(si-NC组vs.si-GFAP组:t=-1.848,P=0.138)的表达无明显影响。结论:在U251细胞中抑制GFAP的表达,影响细胞周期相关蛋白的表达,从而抑制细胞的增殖。
OBJECTIVE: To investigate the effect of GFAP on the proliferation of glioma U251 cells and its possible mechanism by down-regulating the expression of glial fibrillary acidic protein (GFAP) gene by RNA interference (RNAi). Methods: Small interfering RNA (siRNA) was used to specifically interfere with the expression of endogenous GFAP in U251 cells. The m RNA and protein expression of GFAP in U251 cells were detected by RT-PCR and Western blot Level, determine the interference efficiency. The protein expression of GFAP, CREB, cyclin D1, CDK2, CDK4, CDK6, p15, p18, p27, Bax, Bcl-2 and Caspase 3 in U251 cells were detected by Western blot 48 h after transfection with si-NC and si-GFAP MTT assay of cell proliferation in each group. Results: (1) The specific inhibition of GFAP expression in U251 cells inhibited cell proliferation and decreased total cellular total RNA and total protein (si-NC vs si-GFAP group: t = 3.962, P = 0.017; 3.833, P = 0.019). (2) Interference of GFAP expression in U251 cells significantly inhibited CREB (si-NC vs.si-GFAP: t = 4.834, P = 0.008) (t = 3.610, P = 0.023), CDK2 (si-NC group vssi-GFAP group: t = 6.678, P = 0.003) and CDK4 (si- 0.011), and significantly increased the cell cycle-dependent kinase inhibitor p18 (si-NC vs.si-GFAP: t = -3.339, P = 0.029) and p27 GFAP group: t = 13.859, P = 0.000). (3) The results of MTT assay showed that the cell growth rate in si-GFAP group was significantly lower than that in si-NC group at 24 and 48 h (si-NC group vssi-GFAP group: t = 6.389, P = 0.000), the difference was statistically significant. (4) Inhibition of GFAP expression in U251 cells induced apoptosis of Bax (si-NC vs. vs.-GFAP: t = -0.657, P = 0.547) -GFAP group: t = -0.991, P = 0.378) and Caspase 3 (si-NC group vssi-GFAP group: t = -1.848, P = 0.138). Conclusion: The inhibition of GFAP expression in U251 cells affects the expression of cell cycle related proteins and thus inhibits cell proliferation.