miRNA干扰Pokemon基因对大肠癌细胞增殖的影响及机制  

Effect of Pokemon gene on the proliferation of colorectal cancer cells and its mechanism

作　　者：白玉贤[1] 刘磊[1] 隋红[1] 魏孝礼[1] 苑珩珩[1] 

机构地区：[1]哈尔滨医科大学附属第三临床医院肿瘤内科,哈尔滨150081

出　　处：《临床肿瘤学杂志》2011年第11期970-973,共4页Chinese Clinical Oncology

基　　金：黑龙江省教育厅科技研究资助项目(11521179)

摘　　要：目的探索miRNA干扰Pokemon基因后对大肠癌细胞系LoVo增殖和细胞周期的作用。方法构建靶向Pokemon基因的miRNA重组质粒并转染LoVo细胞,设重组质粒组、阴性对照质粒组和脂质体空白对照组,运用实时荧光定量PCR(QPCR)检测干扰后LoVo细胞中Pokemon mRNA的表达,MTT法和流式细胞仪技术分析转染后细胞的增殖和周期变化。结果成功构建含miRNA片段的重组质粒,依次命名为mR22-1、mR22-2、mR22-3和mR22-4;分别转染LoVo细胞后,PokemonmRNA表达水平明显下调,以mR22-1最强,干扰效率为50%。MTT检测显示,mR22-1可明显抑制细胞增殖,24、48和72h的抑制率分别为(23±2.1)%、(47±3.0)%和(69±2.5)%,呈时间依赖性;流式细胞仪测定转染后LoVo细胞周期阻滞在G1期。结论采用RNAi技术可以特异性阻断Pokemon基因的表达;Pokemon基因有促进大肠癌细胞株LoVo增殖的作用。Objective To construct recombinant plasmids containing microRNA targeting the Pokemon gene and investigate the effects of miRNA-mediated downregulation of the Pokemon gene on the proliferation and cell-cycle progression of colorectal cancer LoVo cells.Methods Four miRNAs were designed according to the coding sequence of the Pokemon gene and used to construct recombinant plasmids.The recombinant plasmids were transfected into LoVo cells using Lipofectamine 2000.The expression of Pokemon mRNA was detected by quantitative polymerase chain reaction（QPCR） assay.Cellular proliferation was measured by methyl thiazolyl tetrazolium（MTT） assay.Cell-cycle progression was analyzed by flow cytometry.Results Four recombinant plasmids named mR22-1,mR22-2,mR22-3 and mR22-4 were successfully constructed.The expression of Pokemon mRNA was obviously downregulated in LoVo cells transfected with the recombinant plasmids.The best silencing effect was 50%,which was achieved in cells transfected with the mR22-1 plasmid.MTT assay indicated that mR22-1 transfection could inhibit cellular proliferation.The inhibition rate for 24,48,and 72 hours was（23±2.1）%,（47±3.0）% and（69±2.5）%,and it rose in a time-dependent manner.Flow cytometry assay indicated that mR22-1 transfection led to LoVo cell cycle arrested in G1-phase.Conclusion The recombinant plasmids containing miRNA targeting the Pokemon gene specifically down-regulate Pokemon expression.Pokemon gene can promote proliferation in LoVo cells.

关 键 词：POKEMON基因 大肠癌 RNA干扰 

分 类 号：R735.3[医药卫生—肿瘤]