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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
引用本文:林永敏,任广立,张卫云,蔡启茵,谢聪,马恒颢.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[J].重庆医学,2016(12):1601-1604.
作者姓名:林永敏  任广立  张卫云  蔡启茵  谢聪  马恒颢
作者单位: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
基金项目: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 pmR‐mCherry空质粒组),空白组(未转染组),阳性对照组(HepG2细胞),24 h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72 h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24 h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72 h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。

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

Construction of human microRNA-21 eukaryotic overexpression vector and its up-regulation of c-myc gene expression in HepG2 .2 .15 cells
Lin Yongmin,Ren Guangli,Zhang Weiyun,Cai Qiyin,Xie Cong,Ma Henghao.Construction of human microRNA-21 eukaryotic overexpression vector and its up-regulation of c-myc gene expression in HepG2 .2 .15 cells[J].Chongqing Medical Journal,2016(12):1601-1604.
Authors:Lin Yongmin  Ren Guangli  Zhang Weiyun  Cai Qiyin  Xie Cong  Ma Henghao
Abstract: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 recombi‐nant 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 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was 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 pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;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 h after 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 con‐structed ,this recombinant vector can express miRNA‐21 stably ;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 .
Keywords:microRNAs-21  genetic vectors  genes  c-myc  HBV related liver neoplasms  HepG2  2  15 cells
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