Clusterin基因沉默对脑胶质瘤U87细胞放射敏感性的影响

Effects of gene silencing of Clusterin on radiosensitivity of human glioma U87 cell line

目的:研究通过慢病毒介导的RNA干扰技术沉默簇集蛋白(clusterin,CLU)基因表达对人脑胶质瘤U87细胞放射敏感性的影响。方法:设计和合成靶向CLU的短发夹RNA(short hairprin RNA,shRNA)序列(CLU shRNA1,CLU shRNA2),并构建到慢病毒的载体plentilox 3.7,以不针对任何基因的shRNA序列作为对照(对照shRNA)。包被能成功表达各种shRNA载体的慢病毒。将慢病毒感染人脑胶质瘤U87细胞,Real-time PCR法和Western blot法测定CLU mRNA表达和蛋白质表达水平,证实干扰效果。给予不同放射剂量(2、4、6 Gy)处理后,通过四甲基偶氮唑盐(thiazolyl blue tetrazolium bxomide,MTT)法及流式细胞仪Annexin V/PI法检测脑胶质瘤U87细胞的存活率及凋亡率。Western blot方法筛选多个凋亡相关信号分子的改变。结果:靶向CLU基因的shRNA特异性地抑制了CLU mRNA和蛋白质的表达。MTT实验结果显示实验干扰组细胞在不同放射剂量下的脑胶质瘤实验U87细胞的存活率与对照组比较明显下降,差异具有统计学意义(4 Gy时,对照shRNA vs.CLU shRNA1:P=0.000,对照shRNA vs.CLU shRNA2:P=0.003;6 Gy时,对照shRNA vs.CLU shRNA1:P=0.000,对照shRNA vs.CLU shRNA2:P=0.000)。流式细胞仪Annexin V/PI法检测结果表明:4 Gy放疗后2个实验组细胞凋亡率均明显增加,最高达(35.54±0.95)%,较对照组高2倍多,差异具有明显统计学意义(对照shRNA vs.CLU shRNA1:P=0.000;对照shRNA vs.CLU shRNA2:P=0.000)。Western blot法发现CLU沉默可以显著上调促凋亡分子Bax蛋白水平。结论:CLU基因沉默可能通过调控Bax蛋白的表达,明显增强脑胶质瘤U87细胞对放射的敏感性,有望成为增加脑胶质瘤放射敏感性的新靶点。

Objective：To investigate the effects of gene silencing of Clusterin （CLU） by lentiviurs-mediated RNA interference on radiosensitivity of human glioma U87 cell-line. Methods：shRNAs targeting CLU were cloned into lentiviral vector plentilox3.7 and scramble control shRNA was used as control. Lentiviruses expressing different shRNAs were packaged into 293T cells. Real-time PCR and Western blot were performed to examine mRNA and protein expression of CLU at 72 h after infection of lentivirns. After exposing to different doses of radiation（0,2,4,6 Gy）, MTr assay was used to determine cell survival ratio and apoptotic ratio was also detected by flow cytometry. Changes of apoptotic proteins were examined by Western blot. Results： shRNA targeting CLU specifically inhibited the mRNA and protein level of CLU. MTY results showed that survival ratios of CLU-silencing cells under different doses of ion- izing irradiation（4,6 Gy） were significantly lower compared with those of cells infected with virus expressing control shRNA （4 Gy： control shRNA vs. CLU shRNA1,P=0.000,control shRNA vs. CLU shRNA2,P=0.003;6 Gy：control shRNA vs. CLU shRNA1, P=0.000, control shRNA vs. CLU shRNA2,P=0.000）. Flow cytometry results showed that apoptotic ratios of CLU-silencing cells under 4 Gy treatment were increased to （35.54 ±0.95）%,nearly 2 folds higher than those of control cells（control shRNA vs. CLU shRNA1 ： P=0.000;control shRNA vs. CLU shRNA2：P=0.000）. Results of Western blot showed that gene silencing of CLU markedly upregnlated apoptosis-related protein Bax expression. Conclusions：Gene silencing of CLU significantly enhances the radio-sensitivity of glioma U87 cell-line in vitro partially through upregulating apoptotic protein Bax. CLU may be a promising target during glioma therapy.