中文摘要：

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（P0.05）。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。

英文摘要：

Objective To construct a Luciferace reporter vector containing the 3′untranslated region（3′UTR）of NFAT5 and measure the correlation between NFAT5 and miR-155.Methods The miR-155 targeting NFAT5 3′UTR was predicted by Target Scan,Mir Base and Pic Tar.NFAT5 and mutant NFAT5sequence（NFAT5-mu）were then designed and synthesized,and they were cloned into pMIR-REPORTTM Luciferace reporter vector.Human embryonic kidney-293AD（HEK-293AD）cells of the 4th passage were divided into 4groups according to the random number table.cells in plasimd＋miR-155 mimics groups were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 mimics;cells in plasimd ＋miR-155 mutated groups were transfected with pMIR-NFAT5-mu recombinant plasimid,pRL-Tk plasmid and miR-155mimics;cells in plasimd ＋miR-155 control groups were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 Negative control;cells in plasimd ＋miR-155 inhibitor were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155inhibitor;and were respectively transfected into together by liposome.After culture for 24 h,the luciferase activity was detected by dual luciferase reporter assay system.Results TargetScan,Miranda and PicTar shared the results that NFAT5 has the complementary binding sites with 3′UTR of miR-155.And luciferase reporter vectorwas constructed.Therefore the result of sequencing and double digesting of recombined plasmid were completely correct.Dual-luciferase reporter assay showed that miR-155 possesses a target effect on 3′UTR of NFAT5.Compared to the pMIR-NFAT5＋miR-control group,the luciferase activity of the pMIR-NFAT5＋miR-1 55 mimics group was decreased,with statistically significant difference（P〈0.01）,while there was no significant difference at other time points（P〉0.05）.Conclusion The pMIR-NFAT5 recombinant plasmid and pMIR-NFAT5 recombinant mutated plasmid were confirmed with successful construction.and it was found that miR-155 can target NFAT5 mRNA 3