miR-146a在HepG2.2.15细胞中对c-Myc基因表达影响的研究

摘要

目的 构建has-miR-146a真核过表达载体pmR-146a,探究其在HepG2.2.15肝癌细胞中对c-Myc基因的表达调控作用.方法 PCR扩增has-miR-146a的前体基因片段(pre-has-miR-146a),双酶切后连接到pmR-mCherry载体上,通过菌落PCR、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肝癌细胞中作为实验组,同时设空载体组(转染pmR-mCherry空质粒组),空白组(转染试剂Lipofectamin0 2000+ PBS),24、48 h后观察载体荧光蛋白表达量,qPCR检测各组细胞has-miR-146a表达情况;转染24、48 h后qPCR检测c-Myc基因mRNA表达量,48 h后Western blot检测c-Myc蛋白表达水平.结果 经菌落PCR、双酶切和测序证实,pre-has-miR-146a基因片段插入pmR-mCherry载体中;实验组和空载体组转染24、48 h荧光显微镜观察可见强荧光,与非荧光条件下作对比,转染效率在50％～60％;实验组has-miR-146a表达量明显高于空载体组和空白组(P＜0.01);转染24、48 h后实验组细胞c-Myc基因mRNA表达量较空载体组和空白组低(P＜0.05);转染48 h后,蛋白表达量较空载体组和空白组低(P＜0.01).结论成功构建has-miR-146a真核过表达载体pmR-has-146a,该重组载体可稳定表达has-miR-146a;has-miR-146a可以下调c-Myc癌基因的表达,可以作为治疗原发性肝癌的潜在靶点之一.%Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.