靶向表皮生长因子受体的小分子干扰RNA抑制TJ905人脑胶质瘤细胞增殖和侵袭的实验研究

Inhibitory effects of siRNA targeting epidermal growth factor receptor on proliferation and invasion of human glioblastoma cells

目的 比较靶向表皮生长因子受体 (EGFR)的小分子干扰RNA与反义RNA构建体对TJ90 5人脑胶质瘤细胞增殖和侵袭的抑制作用。方法 选择人EGFR的二个siRNA靶序列构建靶向EGFR的siRNA表达质粒 ,并以反义EGFRRNA为对照进行脂质体介导的TJ90 5人脑恶性胶质瘤细胞系表达。应用免疫荧光和蛋白印迹检查EGFR的表达水平 ;应用TUNEL法分析细胞凋亡 ,流式细胞术分析细胞周期变化 ,MTT法分析细胞增殖 ;蛋白印迹检测基质金属蛋白酶 9的表达 ,明胶酶谱分析MMP9酶活性 ,Transwell法分析侵袭能力。结果 与TJ90 5细胞和转染空载体的TJ90 5细胞比较 ,转染反义EGFRRNA使EGFR表达下调 82 % ,转染siRNA表达质粒组EGFR表达下调 90 %和 92 %。TJ90 5组和空载转染组几乎没有凋亡细胞 ,反义EGFR转染组与siRNA表达质粒组的凋亡率明显增加 ( χ2 =31 5 4 9,P <0 0 0 1) ;siRNA表达载体转染后S期指数较对照和反义RNA转染组细胞明显减少 ,与TJ90 5组和空载转染组比较 ,转染组细胞自第 1天起存活率均明显下降 (P <0 0 0 1) ,且反义组与siRNA表达载体转染组之间存活率差异有显著意义 (P <0 0 1) ;同时蛋白印迹发现转染siRNA表达载体后MMP 9的表达明显下调 ,明胶酶谱分析发现在反义组与siRNA表达载体转染组MMP 9酶活性明显下降 。

Objective To study the inhibitory effects of siRNA targeting epidermal growth factor receptor (EGFR) on the proliferation and invasion of human glioblastoma cells. Method Two siRNA expression constructs using psiRNA NeoG2 vector, that targeted sequences of human EGFR receptor L domain (516 536) and catalytic domain (2400 2420) respectively, were constructed. Human malignant glioma cells of the line TJ905 were cultured in vitro and transfected with pcDNA3 hEGFR, anti sense RNA, blank vector psiRNA NeoG2 (as negative control), psiRNA NeoG2 516, and psiRNA NeoG2 2400 respectively mediated by LipofectAMINE. Immunofluorescence assay and Western blotting were used to detect the EGFR expression. Cell apoptosis was detected by apoptotic index (AI) using TUNEL method. Cell cycle was analyzed by flow cytometry, and cell proliferative activities were measured by MTT. The expression and enzymatic activities of matrix metalloproteinase 9 (MMP 9) were measured by Western blotting and gelatin zymography, and cell invasive capabilities were evaluated by Transwell ECM method. Result Immunofluorescence assay and Western blotting showed that the expression of EGFR was down regulated by 90% and 92% respectively in the siRNA constructs transfected groups, while down regulated by 82% in the antisense EGFR RNA transfected cells in comparison with the TJ905 cells and the cells transfected with blank vector. TUNEL assay showed that almost no apoptotic cell was found in the parental cells or the cells transfected with blank vector, however, apoptosis was increased in antisense EGFR transfected cells (AI=7.2) and siRNA constructs transfected cells (AI=13.7 and 14.7;χ 2=31.549, P <0.001). Flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNA constructs transfected cells than that in the parental cells, the cells transfected with blank vector, and the antisense EGFR transfected cells. MTT assay indicated that compared to the parental cells and the cells transfected with blank vector, the survival rates of transfected cells dramatically dropped down from the first day after implantation ( P <0.05), the siRNA transfected cells demonstrated much lower survival rate than the antisense EGFR transfected cells. Meanwhile, the expression and enzymatic activities of MMP 9 decreased significantly after the transfection of antisense EGFR into the TJ905 cells compared to the TJ905 cells and the cells transfected with blank vector, and were much lower in the siRNA groups than that in the antisense groups ( P <0.05). Cell invasive capability assay demonstrated the similar inhibitory results in the Transwell ECM Matrigel study. ConclusionCompared with antisense approach, siRNA expression constructs targeting EGFR specifically suppresses the EGFR expression, induces gene silencing, and inhibits cell growth and invasion. The plasmid based siRNA approach should be a new strategy in glioma gene therapy targeting EGFR.