miR-146a在HepG2.2.15细胞中对c-Myc基因表达影响的研究

发布时间：2018-10-21 16:55

【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Lipofectamino 2000+PBS),24、48h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、48h后qPCR检测c-Myc基因mRNA表达量,48h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、48h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(P0.01);转染24、48h后实验组细胞c-Myc基因mRNA表达量较空载体组和空白组低(P0.05);转染48h后,蛋白表达量较空载体组和空白组低(P0.01)。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。
[Abstract]:Objective to construct has-miR-146a eukaryotic expression vector pmR-146a, to investigate its role in regulating the expression of c-Myc gene in HepG2.2.15 hepatoma cells. Methods the has-miR-146a precursor gene fragment (pre-has-miR-146a) was amplified by PCR and ligated to the pmR-mCherry vector after double enzyme digestion. The accuracy of the recombinant vector was verified by colony PCR, digestion and sequencing, and the recombinant vector was transfected into HepG2.2.15 hepatoma cells as experimental group. At the same time, empty vector group (transfected with empty pmR-mCherry plasmid group) and blank group (Lipofectamino 2000 PBS), 24 h after transfection) were used to observe the expression of fluorescent protein and the expression of has-miR-146a was detected by qPCR. The expression of c-Myc gene mRNA was detected by qPCR 48 h after transfection, and c-Myc protein was detected by Western blot 48 h later. Results the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector by double enzyme digestion and sequencing of colony PCR, and the strong fluorescence was observed by fluorescence microscope in the experimental group and the empty vector group for 48 h after transfection, and the results were compared with those in the non-fluorescent condition. The expression of has-miR-146a in the experimental group was significantly higher than that in the blank vector group and blank group (P0.01); the expression of c-Myc gene mRNA in the experimental group was lower than that in the empty vector group and blank group after 48 h transfection (P0.05); after 48 h transfection, the protein expression level was lower than that in the empty vector group and blank group (P0.01). Conclusion the successful construction of has-miR-146a eukaryotic expression vector pmR-has-146a, the recombinant vector can stably express has-miR-146a;has-miR-146a can down-regulate the expression of c-Myc oncogene, and can be used as a potential target for the treatment of primary liver cancer.
【作者单位】：广州军区广州总医院儿科;广州中医药大学;广州军区广州总医院检验科;
【基金】：广东省科技项目(2013B03180009) 广东省广州市科技计划项目(201607010123)
【分类号】：R3416

【相似文献】