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

发布时间：2018-02-21 22:10

本文关键词： 微RNA- 遗传载体基因 c-myc 乙型肝炎相关性肝肿瘤 HepG..细胞　出处：《重庆医学》2016年12期 　论文类型：期刊论文

【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(P0.05)。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。
[Abstract]:Objective to construct the eukaryotic expression vector pmR-21 of human minimal RNA-21 microRNA-21 miRNA-21, and to investigate its role in regulating c-myc gene expression in HepG2.2.15 cells. Methods Pre-miRNA-21, a precursor gene fragment of miRNA-21 amplified by PCR, was ligated to pmR-mCherry vector after double enzyme digestion. The accuracy of the recombinant vector was verified by double enzyme digestion and sequencing. The recombinant vector was transfected into HepG2.2.15 cells as experimental group. In addition, the expression of fluorescent protein was observed 24 hours after transfection of pmRmCherry empty plasmid group, blank group (without transfection group, positive control group). Flow cytometry was used to detect transfection efficiency and real-time fluorescence quantitative PCR was used to evaluate the expression of miRNA-21. After 72 hours of transfection, c-myc mRNA and protein expression level were detected by RT-PCR and Western blot. The results showed that the target gene fragment was inserted into the vector by double enzyme digestion and sequencing. The expression of c-myc mRNA in the experimental group was higher than that in the blank vector group 72 hours after transfection, and the cell proliferation in the experimental group was faster than that in the empty vector group and blank group, and the expression of c-myc mRNA in the experimental group was higher than that in the blank vector group. Conclusion miRNA-21 eukaryotic expression vector pmR-21 can stably express miRNA-21 miRNA-21 can up-regulate the expression of c-myc gene and c-myc gene is one of the targets of miRNA-21 for carcinogenesis.
【作者单位】：南方医科大学附属广州军区广州总医院儿科;南方医科大学附属广州军区广州总医院检验科;
【基金】：广州市科技计划基金资助项目(2013J4100116)
【分类号】：R3416

【相似文献】