人microRNA-21真核过表达载体的构建及其在HepG2.2.15细胞中上调c-myc基因的表达  

Construction of human microRNA-21 eukaryotic overexpression vector and its up-regulation of c-myc gene expression in HepG2.2.15 cells

作　　者：林永敏[1] 任广立[1] 张卫云[2] 蔡启茵 谢聪[1] 马恒颢[1] 

机构地区：[1]南方医科大学附属广州军区广州总医院儿科,广州510010 [2]南方医科大学附属广州军区广州总医院检验科,广州510010

出　　处：《重庆医学》2016年第12期1601-1604,共4页Chongqing medicine

基　　金：广州市科技计划基金资助项目(2013J4100116)

摘　　要：目的构建人微小RNA-21(microRNA-21,miRNA-21)真核过表达载体pmR-21,探讨其在HepG2.2.15细胞中对c-myc基因表达的调控作用。方法 PCR扩增miRNA-21的前体基因片段(pre-miRNA-21),双酶切后连接到pmR-mCherry载体上,通过双酶切和测序验证重组载体的准确性;将重组载体转染到HepG2.2.15细胞中为实验组,另设空载体组(转染pmRmCherry空质粒组),空白组(未转染组),阳性对照组(HepG2细胞),24h后观察载体荧光蛋白表达情况,流式细胞术检测转染效率,实时荧光定量PCR评估miRNA-21的表达情况;转染72h后,RT-PCR和Western blot检测c-myc mRNA及蛋白表达水平;CCK-8法检测各组细胞增殖情况。结果经双酶切和测序验证,目的基因片段插入载体中;实验组及空载体组转染24h后细胞内可见强荧光,转染效率大于50%;实验组细胞miRNA-21表达较空载体组、空白组水平升高;转染72h后实验组细胞c-myc mRNA表达较空载体组、空白组升高;实验组细胞增殖快于空载体组及空白组,差异有统计学意义(P<0.05)。结论成功构建miRNA-21真核过表达载体pmR-21,该重组载体可稳定表达miRNA-21;miRNA-21可上调c-myc基因的表达,c-myc基因是miRNA-21发挥促癌作用的靶点之一。Objective To construct the miRNA-21 eukaryotic overexpression vector pmR-21 and to explore its regulation effect on the expression of c-myc gene in HepG2.2.15 cells.Methods The miRNA-21 precursor gene fragment pre-miRNA-21 was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of the recombinant vector was verified by double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells,the fluorescent protein expression was observed under the fluorescence microscopy at 24 hand the transfection efficiency was detected by flow cytometry;the expression of miRNA-21 was evaluated by real-time quantitative PCR;at 72 hafter transfection,the expression levels of c-myc gene were detected by RT-PCR and Western blot;CCK-8was used to detect the cell proliferation in each group.Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmRmCherry vector;at 24 hafter transfection,intracellular strong fluorescence was seen,the transfection efficiency was higher than50%;miRNA-21 expression level of the pmR-21 recombinant vector group was significantly increased;c-myc gene expression was increased in the pmR-21 recombinant vector group at 72 hafter transfection,the cell proliferation in the pmR-21 recombinant group was faster than that in the control group（P〈0.05）.Conclusion The pmR-21 eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miRNA-21stably;miRNA-21 can up-regulate c-myc gene expression,c-myc gene is one of miR-21′s targets for playing a cancer-promoting action.

关 键 词：微RNA-21 遗传载体 基因 C-MYC 乙型肝炎相关性肝肿瘤 HEPG2.2.15细胞 

分 类 号：R346[医药卫生—基础医学]