PTTG靶向siRNA表达载体构建及其沉默效率评价

目的：构建针对人脑胶质瘤细胞系U251的高效率沉默垂体瘤转化基因(PTTG)的小分子干扰RNA(siRNA)表达载体。 方法：合成特异性干扰PTTG 的siRNA片段，并与pGenesil2 载体连接，构建PTTG干扰载体（pGenesil2-PTTG siRNA）。利用脂质体将其转染U251细胞，分为正常细胞对照组、HK阴性对照组和3个siRNA干扰组（pGenesil2-PTTG siRNA1、pGenesil2-PTTG siRNA2和pGenesil2-PTTG siRNA3），应用半定量逆转录聚合酶链式反应(RT-PCR)法和流式细胞术对转染后U251细胞中PTTG 的mRNA和蛋白表达水平进行分析。 结果：酶切证实PTTG-siRNA表达载体构建成功，插入片段测序结果与合成的siRNA结果一致；转染pGenesil2-PTTG siRNA后，3个干扰组的U251 细胞中PTTG基因和蛋白表达水平均较正常对照组显著降低（P<0.01）。 结论：成功构建了能高效率沉默PTTG 的RNAi表达载体；pGenesil2-PTTG siRNA高效地抑制了胶质瘤U251细胞中PTTG基因的表达。

Construction of PTTG siRNA expressing vector and evaluation of its gene silencing efficiency

Objective To construct the specific high efficiency small interfering RNA(siRNA) expression vector that can silence PTTG gene.Methods Using vector based RNA interference technique,vectors were constructed to transcribe functional siRNA specially targeting PTTG.The vectors were used to transfect U251 cells by lipofectmine2000 reagent.And the cells were dividedinto five groups: normal control,HK negative group and three siRNA interfering groups(pGenesil2-PTTG siRNA1,pGenesil2-PTTG siRNA2 and pGenesil2-PTTG siRNA3). The expression levels of mRNA and protein of PTTG were analyzed by RT-PCR and flow cytometry methods.Results By restriction endonuclease and DNA sequencing analyzing,eukyaryotic expression plasmid of PTTG was successfully constructed.PTTG mRNA and its protein level in transfected U251 cells with PTTG RNAi plasmid decreased significantly compared with the normal control group(P<0.01).Conclusion pGenesil2-PTTG siRNA vectors were successfully constructed,and they can silence the PTTG gene in U251 cells with high efficiency.