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目的探讨短发夹核糖核酸(sh RNA)对高表达EZH2的人胶质瘤细胞株U251增殖和侵袭的影响。方法构建针对EZH2的sh RNA重组质粒,采用电穿孔的方法转染至U251细胞中,分为对照组、control-sh RNAGFP空载体转染组和EZH2-sh RNA重组质粒转染组。分别通过逆转录-聚合酶链反应和蛋白质印迹法检测各组细胞EZH2基因和蛋白的表达,以噻唑蓝法测各组细胞的增殖活性,蛋白质印迹法检测各组细胞的EZH2蛋白表达水平;Transwell小室检测细胞侵袭力。结果对照组、control-sh RNA-GFP空载体转染组、EZH2-sh RNA重组质粒转染组U251细胞EZH2基因的表达分别为1.13±0.05、1.15±0.05、0.19±0.02,U251细胞EZH2蛋白的表达分别为1.03±0.03、0.97±0.06、0.20±0.02,转染后24 h细胞增殖能力分别为100.00%±9.31%、100.03%±9.35%、60.13%±3.15%,转染后48 h细胞增殖能力分别为100.00%±9.13%、99.58%±9.27%、53.01%±3.14%,重组质粒转染组较另两组均明显下降,差异有统计学意义(P<0.05);重组质粒转染组转染48 h后细胞磷酸化蛋白激酶B和磷酸化叉头框蛋白O1水平降低、细胞周期蛋白D1表达减少、p27蛋白表达增多,差异有统计学意义(P<0.05)。Transwell小室侵袭实验显示侵袭至Transwell小室滤膜下表面的细胞数在EZH2-sh RNA重组质粒转染组[(46.00±2.82)个]低于对照组[(60.67±5.71)个]和control-sh RNA-GFP空载体转染组[(61.00±2.48)个],差异有统计学意义(P<0.01)。结论 EZH2基因沉默能有效抑制人胶质瘤细胞的增殖和侵袭能力,为深入研究EZH2在胶质瘤中作用提供了研究基础。
Objective To investigate the effect of short hairpin RNA (shRNA) on the proliferation and invasion of human glioma cell line U251 with high expression of EZH2. Methods The sh RNA recombinant plasmid targeting EZH2 was constructed and transfected into U251 cells by electroporation. The recombinant plasmid was divided into control group, control-sh RNAGFP empty vector transfection group and EZH2-sh RNA recombinant plasmid transfection group. The expression of EZH2 gene and protein in each group was detected by reverse transcription-polymerase chain reaction and Western blot respectively. The proliferation activity of each group was detected by thiazolyl blue. The expression of EZH2 protein was detected by Western blotting. Transwell Cell detection of cell invasiveness. Results The expression of EZH2 gene in U251 cells transfected with control-sh RNA-GFP vector was 1.13 ± 0.05, 1.15 ± 0.05 and 0.19 ± 0.02, respectively. The expression of EZH2 in U251 cells The expression levels were 1.03 ± 0.03, 0.97 ± 0.06 and 0.20 ± 0.02, respectively. The proliferation of cells at 24 h after transfection were 100.00% ± 9.31%, 100.03% ± 9.35% and 60.13% ± 3.15%, respectively. (100.00 ± 9.13%, 99.58% ± 9.27% and 53.01% ± 3.14%, respectively). The transfected group showed a significant decrease compared with the other two groups (P <0.05). The recombinant plasmids transfected group After 48 h of transfection, the levels of phosphorylated protein kinase B and phosphorylated FHP O1 were decreased, the expression of cyclin D1 and the expression of p27 protein were increased, with statistical significance (P <0.05). Transwell chamber invasion assay showed that the number of cells invaded to the lower surface of Transwell chamber was significantly lower in EZH2-sh RNA transfection group (46.00 ± 2.82) than in control group (60.67 ± 5.71) and control-sh RNA-GFP empty vector transfection group [(61.00 ± 2.48)], the difference was statistically significant (P <0.01). Conclusion EZH2 gene silencing can effectively inhibit the proliferation and invasion ability of human glioma cells, providing the basis for further study on the role of EZH2 in glioma.