靶向Pokemon基因shRNA重组质粒的构建及其对HepG2细胞生长的影响  

Short hairpin RNA-mediated downregulation of the Pokemon gene suppresses proliferation and promotes apoptosis in HepG2 cells

作　　者：王波[1] 张全乐[1] 晏维[1] 夏丽敏[1] 刘梅[1] 田德安[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院消化内科,湖北省武汉市430030

出　　处：《世界华人消化杂志》2009年第30期3128-3133,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30800968~~

摘　　要：目的:观察携带shRNA的重组质粒对肝癌细胞HepG2增殖及凋亡的影响,并初步探讨其分子机制.方法:设计3对针对Pokemon基因不同位点的shRNA片段的真核表达载体,脂质体介导转染肝癌细胞株HepG2细胞.RT-PCR及Western blot检测转染前后Pokemon mRNA及蛋白质的表达,MTT和流式细胞仪分析转染后细胞增殖及凋亡的变化,RT-PCR检测其可能的下游因子β-catenin及H-ras的表达.结果:成功构建含shRNA片段的重组质粒,依次命名为pshRNA1、2、3.转染HepG2细胞后,Pokemon基因的mRNA及蛋白质表达水平明显下调,以pshRNA2最强,抑制率分别为75.2%(RNA水平)和72.61%(蛋白质水平).MTT显示pshRNA2可明显抑制细胞增殖;流式细胞仪测定转染后细胞凋亡增加;RT-PCR显示下游因子H-ras的表达明显降低(P<0.05),而β-catenin的表达没有变化(P>0.05).结论:采用RNAi技术可以特异性阻断Pokemon基因的表达;Pokemon基因有促进肝癌细胞株增殖及抑制其凋亡的作用,该效应可能与下调其下游因子H-ras的表达有关.AIM： To construct recombinant plasmids containing short hairpin RNA （shRNA） targeting the Pokemon gene and investigate the effects of shRNA-mediated downregulation of the Pokemon gene on the proliferation and apoptosis of HepG2 cells. METHODS： Three shRNAs were designed according to the coding sequence of the Pokemon gene and used to construct recombinant plasmids. The recombinant plasmids were trans-fected into HepG2 cells using Lipofectamine 2000. The expression of Pokemon mRNA and protein was detected by reverse transcriptionpolymerase chain reaction （RT-PCR） and Western blot, respectively. Cellular proliferation was measured by methyl thiazolyl tetrazolium （MTT） assay. Apoptosis was analyzed by flow cytometry. The expression of H-ras and β-catenin genes was detected by RT-PCR. RESULTS： Three recombinant plasmids were successfully constructed. The expression of Pokemon mRNA and protein was obviously downregulated in HepG2 cells transfected with the recombinant plasmids. The best silencing effect was achieved in cells transfected with the pshRNA2 plasmid. The expression levels of Pokemon mRNA and protein were downregulated by 75.2% and 72.61%, respectively. MTT assay indicated that pshRNA2 transfection could inhibit cellular proliferation and promote apoptosis. After pshRNA2 transfection, the expression of H-ras mRNA was downregulated （P〈0.05） in HepG2 cells though no signifi cant change was observed in β-catenin expression. CONCLUSION： The recombinant plasmids containing shRNA targeting the Pokemon gene can specifically downregulate Pokemon expression. The Pokemon protein can promote proliferation and inhibit apoptosis in HepG2 cells possibly via downregulation of H-ras expression.

关 键 词：POKEMON 短发夹状RNA 细胞增殖 细胞凋亡 H-RAS 

分 类 号：R735.7[医药卫生—肿瘤]