靶向PERK基因的shRNA真核表达载体的构建及对内质网应激状态下L02肝细胞凋亡的影响

Construction of shRNA eukaryotic expression vectors targeting PERK gene and effect of PERK gene knockdown on apoptosis in endoplasmic reticulum stress-induced L02 hepatocytes

目的:构建靶向RNA依赖的蛋白激酶样内质网激酶(PERK)基因的短发夹状RNA(shRNA)真核表达载体,观察其对内质网应激(ERS)状态下人正常肝细胞株L02凋亡的影响。方法:根据PERK基因核苷酸序列,按照小干扰RNA(siRNA)靶序列的设计原则,设计并构建靶向PERK基因mRNA的3个特异性shRNA表达载体(PERK1-shRNA、PERK2-shRNA、PERK3-shRNA)和1个无同源性的阴性对照载体(HK-shRNA),经酶切和测序鉴定确认shRNA载体构建成功后,转染L02肝细胞,RT-PCR和Western blotting方法检测PERK基因的表达,筛选出抑制效果最佳的表达载体。分别应用MTT法和流式细胞术检测ERS状态下沉默了PERK基因的L02肝细胞活力和凋亡的变化。结果:经酶切和测序鉴定证实,4个shRNA表达载体均构建成功。RT-PCR和Westernblotting结果显示,与空白对照组相比,PERK1-shRNA、PERK2-shRNA、PERK3-shRNA组L02细胞中PERK基因在mRNA及蛋白水平上的表达均明显降低(P<0.05),其中PERK1-shRNA干扰效果最强。MTT法和流式细胞术结果表明,沉默PERK基因能明显增加ERS状态下肝细胞活力、抑制其凋亡(P<0.05)。结论:成功构建靶向PERK基因的shRNA真核表达载体,PERK基因缺失对ERS状态下L02肝细胞凋亡有抑制作用。

AIM： To construct short hairpin RNA（shRNA） eukaryotic expression vectors targeting the gene of RNA-dependent protein kinase（PKR）-like endoplasmic reticulum kinase（PERK）,and to observe the effect of PERK gene knockdown on apoptosis of human normal hepatic L02 cells treated with thapsigargin.METHODS： Three shRNA expression vectors targeting PERK gene,named PERK1-shRNA,PERK2-shRNA and PERK3-shRNA,and one non-homologous negative control expression vector（HK-shRNA） were constructed based on the nucleotide sequence of PERK and the criteria of designing small interfering RNA（siRNA）,and were identified by enzyme digestion and DNA sequencing analysis.After L02 hepatocytes were transfected with the plasmids,the PERK expression was determined by RT-PCR and Western blotting,and the plasmid with the best inhibitory effect on PERK expression was screened.The cell viability and apoptotic rate of L02 hepatocytes transfected with PERK-shRNA under endoplasmic reticulum stress（ERS） were measured by the methods of MTT and flow cytometry,respectively.RESULTS： Four shRNA expression vectors were constructed.The mRNA and protein expression levels of PERK gene decreased significantly in L02 hepatocytes transfected with PERK1-shRNA,PERK2-shRNA and PERK3-shRNA as compared with those in control cells（P0.05）.The interfering effect of PERK1-shRNA on PERK gene expression was the best.PERK knockdown by PERK1-shRNA increased the viability and inhibited the apoptosis of L02 cells under ERS.CONCLUSION： The shRNA expression vectors targeting PERK gene are constructed,and PERK gene knockdown may inhibit apoptosis in L02 hepatocytes under ERS.