靶向XIAP的shRNA重组质粒的构建

Construction of recombinant plasmids containing short hairpin RNA targeting the X-linked inhibitor of the apoptosis protein gene

目的:设计以X染色体连锁凋亡抑制蛋白(XIAP)为靶向的shRNA,构建携带此shRNA的重组质粒,检测其抑制XIAP表达的效应,筛选RNA干扰作用最强的shRNA片段.方法:设计4对针对XIAP基因不同位点的shRNA片段,构建携带此shRNA片段的真核表达载体psiRNA-Hhneo-XIAP,通过脂质体介导的方法将重组质粒转染到肝癌细胞株HepG2细胞中.采用逆转录酶链式反应(RT-PCR)及蛋白印迹(Western blotting)方法检测XIAP的mRNA及蛋白表达情况,比较转染前后其表达差异,以判断各shRNA的干扰效应.结果:成功构建含shRNA片段的重组质粒.经质粒测序证实,插入的DNA片段的序列与设计序列完全一致.重组质粒转染HepG2细胞后,XIAP基因的mRNA水平及蛋白表达水平明显下调,其中以1号重组质粒效应最强.shRNA作用48h后,对HepG2细胞中XIAP mRNA和蛋白的抑制率与3,4号重组质粒相比,均具有显著性差异(mRNA:94.5% vs 81.5%,82.6%,均P<0.01:蛋白:92.6% vs 80.7%,82.9%,均P<0.01).结论:成功构建了携带以XIAP为靶向的shRNA的重组质粒,其对肝癌细胞内XIAP的表达具有显著抑制效应.<正>X染色体连锁凋亡抑制蛋白;;短发夹状RNA;;肝癌;;逆转录聚合酶链式反应;;

AIM： To construct recombinant plasmids containing short hairpin RNA （shRNA） that targets the X-linked inhibitor of apoptosis protein （XIAP） gene, to assay the expression of XIAP in HepG2 cells after transfecting with recombinant plasmids, and to detect the RNAi effect of shRNA. METHODS： Four pairs of shRNAs that target the XIAP gene were designed. The eukaryotic expression vector of psiRNA-Hhneo-XIAP was constructed and transfected via liposomes into HepG2 cells. XIAP mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction. XIAP protein expression was examined by Western blotting. The difference in XIAP gene and protein expression levels between cells transfected or not transfected by psiRNA- Hhneo-XIAP was compared. RESULTS： The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. Transfection of psiRNA-Hhneo-XIAP significantly down-regulated XIAP expression in HepG2 cells. Recombinant plasmid 1 had the strongest effect, with an inhibition ratio of 94.5% at the mRNA level and 92.6% at the protein level, which showed a significant difference from plasmids 3 and 4 （mRNA： 94.5% vs 81.5%, 82.6%, both P 〈 0.01; protein： 92.6% vs 80.7%, 82.9%, both P 〈 0.01）. CONCLUSION： XIAP gene targeted shRNA suppresses gene and protein expression.