RNA 干扰抑制 EZH2基因表达对人胶质瘤细胞凋亡及对卡莫司汀敏感性的影响

摘要

目的：观察RNA干扰技术抑制EZH2基因表达对人胶质瘤U251细胞凋亡及其对卡莫司汀敏感性的影响。方法针对EZH2 mRNA序列设计合成小干扰RNA（ siRNA）的DNA模板，构建pRNAT-EZH2重组表达载体，并用其转染人胶质瘤U251细胞。用RT-PCR法检测pRNAT-EZH2转染前后U251细胞中内源性EZH2表达；激光共聚焦显微镜观察转染后及加入卡莫司汀后U251细胞凋亡情况；MTT法检测转染前后U251细胞对化疗药物卡莫司汀敏感性；用分光光度法检测转染前后以及加入卡莫司汀后U251细胞中Caspase-3活性。结果成功构建pR-NAT-EZH2重组质粒，并成功转染U251细胞。 pRNAT-EZH2重组质粒转染后U251细胞中EZH2 mRNA表达量降低（ P＜0．01）；卡莫司汀组及卡莫司汀加pRNAT-EZH2组U251细胞凋亡明显；与卡莫司汀联合时，pRNAT-EZH2组U251细胞生长抑制率明显增高（P＜0．01）；Caspase-3活性明显高于空白对照组和阴性对照组（P均＜0．01）。结论 pRNAT-EZH2可抑制EZH2在人胶质瘤U251细胞中的表达，并增强U251细胞对卡莫司汀敏感性。%Objective To explore the relationship between the expression of EZH2 and BCNU Chemosensitivity and Apoptosis of human glioma U251 cells by decreasing EZH2 gene expression by RNA interference.Methods One pair of DNA template coding siRNA was synthesized against U251 to reconstruct pRNA-EZH2, which was transfected into U251 cells.The EZH2 expression in U251 cells were transfected with pRNAT-EZH2, cells were exposed to BCNU and it was de-tected by RT-PCR.MTT was used to observe the growth inhibiting ratio induced by BCNU in U251 cells.Cell apoptosis was analyzed by fluorescence microscopy.Caspase-3 activity was measured by spectrofluorometer.Results RT-PCR ana-lyses demonstrated that pRNAT-EZH2 could significantly inhibit the expression of EZH2 in U251 cells (P<0.01); after U251 cells were exposed to BCNU, the growth inhibiting ratio, Caspase-3 activity and cell apoptosis of pRNAT-EZH2 transfected cells was significantly increased compared to that of control group and negative group (P<0.01).Conclusion pRNAT-EZH2 can significantly inhibit the expression of EZH2, increase chemosensitivity to BCNU and apoptosis of U251 cells.