人MicroRNA335的预测靶基因CCL11 CCL26及SOX4的鉴定  

The identification of human microRNA335′s predicated target gene CCL11,CCL26 and SOX4

作　　者：温志红[1] 代艳[1] 何爽[2] 

机构地区：[1]广西壮族自治区人民医院儿科,南宁530021 [2]广西壮族自治区人民医院康复科,南宁530021

出　　处：《重庆医学》2015年第24期3316-3318,共3页Chongqing medicine

基　　金：广西自然科学基金项目(2011GXNSFC018019);广西卫生和计划生育委员会计划课题项目(Z2015309)

摘　　要：目的验证人MicroRNA335(hsa-miR-335)与嗜酸细胞趋化因子1(CCL11)、嗜酸细胞趋化因子3(CCL26)、SOX4的靶向调控关系。方法选择网络在线的生物信息学软件miRanda和TargetScan共同预测为hsa-miR-335靶基因的CCL11、CCL26、SOX4作为研究对象。构建CCL11、CCL26、SOX4基因3′端非翻译区(3′UTR)双荧光素酶报告基因载体pMIR-REPORT-CCL11 3′UTR、pMIR-REPORT-CCL26 3′UTR、pMIR-REPORT-SOX4 3′UTR;采用Lipofectamine 2000将真核表达质粒pMIR-REPORT-CCL11 3′UTR、pMIR-REPORT-CCL26 3′UTR、pMIR-REPORT-SOX4 3′UTR、阳性对照质粒分别与Pre-miRTM miRNA335Precursor或阴性对照质粒共转染到293T7/17细胞;双荧光素酶报告基因检测系统检测CCL11、CCL26、SOX4基因3′UTR与Hsa-mir-335共转染萤火虫(Firefly)和海洋腔肠(Renilla)荧光素酶活性,并与阴性对照对比。结果hsa-miR-335对SOX4的荧光表达有显著的抑制作用(P<0.01),但并不影响CCL11、CCL26荧光素酶活性(P>0.05)。结论 hsa-miR-335可靶向调控SOX4,而对CCL11、CCL26的表达无影响。Objective To identify the targeted-regulating relationship between human MicroRNA335 （hsa-miR-335）and CCL11,CCL26 and SOX4. Methods The potential fragments of hsa-miR-335 target genes CCLll, CCL26 and SOX4 were predicted by the bioinformatics analyzing tools online. The 3＇ untranslated regions（3~UTR） of the CCL11 ,CCL26 and SOX4 were connected to the eukaryotic expression vectors pMIR REPORT. The constructs of pMIR-REPORT-CCL113~UTR, pMIR-REPORT-CCL26 3＇ UTR,pMIR-REPORT-SOX4 3＇UTR and positive control were co-transfected with Pre-miRTM miRNA335 Precursor or negative control into 293 T7/17 cell line by lipofectamine 2000 ,respectively. Both Firefly and Renilla luciferase activity were detected by dual luciferase reporter assay system. Results Compared with the negative control group,luciferase assay revealed that has-miR -335 could significantly diminish lueiferase activity from SOX4 reporter vector （P^0.01） , while the suppression of luciferase activity was not found in CCL11 or CCL26 reporter vector （P〈0.05）. Conclusion The results suggested that hsa-miR-335 targeted regu- lated SOX4,but not targeted CCLll and CCL26.

关 键 词：微RNAS hsa-miR-335 靶基因 基因调控 CCL11 CCL26 SOX4 双荧光素酶报告基因检测系统 

分 类 号：Q78[生物学—分子生物学]