SOCS3基因3'端非翻译区荧光素酶报告载体的构建及活性鉴定

Construction of SOCS3 3′-UTR luciferase reporter vector and identification of its activity

目的:研究细胞因子信号抑制蛋白-3(suppressors of cytokine signaling-3,SOCS3)基因3'端非翻译区(3'-un-translated region,3'-UTR)微小RNA(microRNA,miRNA)的调控作用,构建重组SOCS3基因3'-UTR荧光素酶报告载体,并通过荧光素酶活性测定,初步分析可能调控SOCS3基因表达的miRNAs。方法:采用PCR方法从原代培养的小鼠星形胶质细胞基因组DNA中扩增SOCS3基因3'-UTR序列,插入荧光素酶报告载体pGL3-Promoter,获得重组载体pGL3-Promoter/SOCS3;通过Target Scan5.2、RNAhybrid和FINDTAR3软件预测可能与SOCS3基因3'-UTR结合的miR-NAs;将pGL3-Promoter/SOCS3重组质粒和miRNAs共转染HEK 293T细胞,测定SOCS3 3'-UTR荧光素酶的活性。结果:酶切及核酸测序证实,成功构建了SOCS3基因3'-UTR序列的荧光素酶报告重组子;miRNA靶位点预测显示,SOCS3基因可能是miR-203、miR-291a-5p、miR-9、miR-140和miR-130b的作用靶标;与对照组相比,miR-203、miR-291a-5p、miR-140能使SOCS3 3'-UTR荧光素酶的活性显著降低。结论:成功构建了SOCS3基因3'-UTR荧光素酶报告载体,且miR-203、miR-291a-5p、miR-140可显著降低其荧光素酶的活性。

Objective： To study the microRNA（miRNA） targeting to 3′-untranslated region（3′-UTR） of suppressors of cytokine signaling-3（SOCS3） gene,a SOCS3 3′-UTR luciferase reporter vector was constructed and to analyse the miRNAs regulated SOCS3 expression through detecting the luciferase activity of SOCS3 3′-UTR.Methods： The 3′-UTR fragment of SOCS3 gene was amplified by PCR from genomic DNA of primary astrocytes of mouse and cloned into luciferase reporter vector（pGL3-Promoter）,then the recombinant pGL3-Promoter/SOCS3 was identified.The miRNAs targeting SOCS3 3′-UTR was predicted by Target Scan5.2,RNAhybrid and FINDTAR3 softwares.Thereafter,pGL3-Promoter/SOCS3 and miRNAs were co-transfected into HEK 293T cells and the luciferase activity of SOCS3 3′-UTR was measured.Results： 3′-UTR fragment of SOCS3 gene was successfully cloned into the pGL3-Promoter reporter vector,which verified by Xba I digestion and DNA sequencing.The predicted miRNAs targeting SOCS3 3′-UTR included miR-203,miR-291a-5p,miR-9,miR-140 and miR-130b.Compared with the control group,the luciferase activity of pGL3-Promoter/SOCS3 treated with miR-203,miR-291a-5p,or miR-140 was remarkably decreased,respectively.Conclusion： The SOCS3 3′-UTR luciferase reporter vector was constructed successfully,and the luciferase activity of the recombinant vector can be suppressed significantly by miR-20,miR-291a-5p or miR-140.