p53基因3′UTR双荧光素酶报告质粒的构建及其与miRNA-2127靶向关系的验证
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摘要
为了验证miRNA-2127与p53基因的靶向关系,利用生物信息学软件预测p53与miRNA-2127的结合位点,全基因合成法合成该结合位点的野生型与突变型模板,并将其克隆到pmiR-RB-REPORT~(TM)双荧光素酶报告载体中,构建野生型与突变型重组双荧光素酶报告质粒。将293T细胞分为4组,分别共转染野生型报告质粒+阴性对照(NC)、野生型报告质粒+miRNA-2127、突变型报告质粒+NC、突变型报告质粒+miRNA-2127。检测各组细胞中荧光素酶活性差异,结果显示:共转染了野生型报告质粒+miRNA-2127的293T细胞与共转染了野生型报告质粒+NC组相比,荧光素酶活性显著降低(P<0.05);且突变型报告质粒+miRNA-2127组的相对荧光素酶活性显著高于野生型报告质粒+miRNA-2127(P<0.05),说明miRNA-2127能够靶向调控p53基因,且结合位点位于3′UTR区369—375间。
To verify the targeted relationship between miRNA-2127 and p53, the binding sites of p53 and miRNAs-2127 were predicted by bioinformatics software. The wild-type and mutant templates of the binding sites were synthesized by the whole gene synthesis method, and were cloned into pmiR-RB-REPORT~(TM) dual-luciferase reporter vector to construct the wild-type and mutant-type recombinant dual-luciferase reporter plasmids. The cultured monolayer 293 T cells were divided into 4 groups including co-transfected wild-type reporter plasmid + NC control, co-transfected wild-type reporter plasmid + miRNA-2127, co-transfected mutant reporter plasmid + NC control and co-transfected mutant reporter plasmid + miRNA-2127, respectively. The difference of luciferase activity in each group was detected. The results showed that compared with co-transfected wild-type plasmid + NC control, the relative luciferase activity of 293 T cells in the group with co-transfected wild-type plasmid + miRNA-2127 showed a significant decrease(P<0.05), and that of co-transfected mutant reporter plasmid + miRNA-2127 group was significantly higher than co-transfected wild-type reporter plasmid + miRNA-2127 group. The results indicated that miRNA-2127 could targeted regulate p53 and the binding sites were located between 369 and 375 in the 3′ UTR region.
To verify the targeted relationship between miRNA-2127 and p53, the binding sites of p53 and miRNAs-2127 were predicted by bioinformatics software. The wild-type and mutant templates of the binding sites were synthesized by the whole gene synthesis method, and were cloned into pmiR-RB-REPORT~(TM) dual-luciferase reporter vector to construct the wild-type and mutant-type recombinant dual-luciferase reporter plasmids. The cultured monolayer 293 T cells were divided into 4 groups including co-transfected wild-type reporter plasmid + NC control, co-transfected wild-type reporter plasmid + miRNA-2127, co-transfected mutant reporter plasmid + NC control and co-transfected mutant reporter plasmid + miRNA-2127, respectively. The difference of luciferase activity in each group was detected. The results showed that compared with co-transfected wild-type plasmid + NC control, the relative luciferase activity of 293 T cells in the group with co-transfected wild-type plasmid + miRNA-2127 showed a significant decrease(P<0.05), and that of co-transfected mutant reporter plasmid + miRNA-2127 group was significantly higher than co-transfected wild-type reporter plasmid + miRNA-2127 group. The results indicated that miRNA-2127 could targeted regulate p53 and the binding sites were located between 369 and 375 in the 3′ UTR region.
引文
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[2] 李宇鹏, 张一鸣, 胡海碧,等. 肝癌细胞HepG2中p53调控miRNA-3661的生物信息分析与功能验证[J]. 生物技术通报, 2017, 33(7):216-223.
[3] Le M T N, Teh C, Shyh-Chang N, et al. MicroRNA-125b is a novel negative regulator of p53 [J].Genes & Development, 2009, 23(7): 862-876.
[4] Hermeking H. MicroRNAs in the p53 network: micromanagement of tumour suppression [J].Nature Reviews Cancer, 2012, 12(9): 613-626.
[5] 魏斌, 邓霞, 卞秀娟,等. AKT2 3′-UTR荧光素酶报告基因载体构建及与miRNA-625靶向关系验证[J]. 江苏大学学报(医学版),2016, 26(3):204-207.
[6] 贾春平. 抑癌基因p53与肿瘤研究的最新进展[J]. 生命科学, 2008, 20(3):450-453.
[7] Takaoka A, Hayakawa S, Yanai H, et al. Integration of interferon-α/β signalling to p53 responses in tumour suppression and antiviral defence [J]. Nature, 2003, 424(6948): 516-523.
[8] Fábián Z, Csatary C M, Szeberényi J, et al. p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines [J]. Journal of Virology,2007, 81(6): 2817-2830.
[9] 欧阳伟, 王永山, 王永强,等. 传染性法氏囊病病毒感染细胞内源性microRNA表达差异分析[J]. 中国兽医学报, 2012, 32(3):329-336.
[10] Voorhoeve P M, le Sage C, Schrier M,et al. A genetic screen implicates miRNA-372 and miR-373 as on cogenes in testicular germ cell tumors[J]. Cell, 2006, 124(6):1169-1181.