NFAT5基因报告载体的构建及其与miR-155关系的实验研究

发布时间：2018-03-04 11:29

本文选题：T淋巴细胞核因子　切入点：miR-　出处：《重庆医学》2017年08期 　论文类型：期刊论文

【摘要】：目的通过生物信息软件预测出miR-155的靶基因,构建miR-155靶基因荧光素酶基因报告载体,验证其与miR-155的对应关系。方法以数据库miR Base为依据,对miR-155进行生物信息学分析,通过Target Scan、Mir Base、Pic Tar三大软件预测miR-155的靶基因;将设计合成的T淋巴细胞核因子5(NFAT5)及其突变序列NFAT5-mu序列分别克隆至荧光素酶报告质粒pMIR-REPORT~(TM) Luciferace;将第4代人胚胎肾293AD(HEK-293AD)细胞按随机数字表法分为4组:miR-155mimics+pMIR-NFAT5组、miR-155 mimics+pMIR-NFAT5-mu组、miR-155inhibitor+pMIR-NFAT5组、miR155 Negative control+pMIR-NFAT5组与对照质粒(pRL-TK)共转染24h后用双荧光素酶检测试剂盒测定相对荧光素酶活性。结果 Mirbase、TargetScan、PicTar预测交叉结果显示,NFAT5基因3′UTR与miR-155存在结合互补位点;构建pMIR-NFAT5、pMIR-NFAT5-mu重组质粒经酶切及测序鉴定正确;双荧光素酶报告基因检测系统显示:pMIR-NFAT5+miR155 mimics组较pMIR-NFAT5+miR-control组荧光素酶活性降低,差异有统计学意义(P0.01);而其他组间差异无统计学意义(P0.05)。结论成功构建pMIR-NFAT5、pMIR-NFAT5-mu荧光素酶报告基因重组质粒;证实miR-155对NFAT5基因mRNA 3′-UTR具有靶向调控作用,为下一步研究miR-155在吸入性肺损伤机制中的作用提供前期实验室数据和方法。
[Abstract]:Objective to predict the target gene of miR-155 by bioinformatics software, construct the luciferase gene report vector of miR-155 target gene, and verify the corresponding relationship with miR-155. Methods based on the miR Base database, the bioinformatics analysis of miR-155 was carried out. The target genes of miR-155 were predicted by three software of Target Scan-Mir base Tar. The synthesized T lymphocyte nuclear factor 5 (NFAT5) and its mutated NFAT5-mu sequence were cloned into luciferase reporter plasmid pMIR-REPORTT) Luciferace.The fourth generation of human embryonic kidney 293ADHEK-293ADcells were divided into 4 groups according to random number table: miR-155mimics pMIR-NFAT5 group miR-155 mimics pMIR-NFAT5-mu group miR-155 mimics pMIR-NFAT5-mu group miR-155inhibitor pMIR-NFAT5. Two luciferase assay kit was used to detect the relative luciferase activity after 24 hours of co-transfection with control plasmid pRL-TK. results Mirbase TargetScann PicTar predicted the crossover of Negative pMIR-NFAT5 and pRL-TK. the results showed that there were complementary binding sites between 3UTR and miR-155 of NF-AT5 gene. The recombinant plasmid pMIR-NFAT5-mu was constructed and confirmed by restriction endonuclease digestion and sequencing. The detection system of double luciferase reporter gene showed that the luciferase activity in the miR155 mimics group was lower than that in the pMIR-NFAT5 miR-control group. Conclusion the recombinant plasmid of pMIR-NFAT5pMIR-NFAT5-mu luciferase reporter gene was successfully constructed, and it was proved that miR-155 has targeted regulation effect on NFAT5 gene mRNA 3H-UTR. To provide experimental data and methods for the further study of the role of miR-155 in the mechanism of inhalation lung injury.
【作者单位】：河北北方学院研究生部;解放军第三○九医院烧伤整形科;
【基金】：国家自然科学基金面上资助项目(81372051,81670009) 总参军事医学和老年病科研基金项目(ZCWS14B06) 解放军309医院院管课题(2014MS-001) 北京市科技计划“首都特色”专项基金资助项目(Z151100004015199) 全军医学科技青年培育项目(15QNP049)
【分类号】：R563

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