微小RNA-181a在人食管癌EC9706细胞中对靶基因PRDM1调控作用的研究  

The regulation effects of miR-181a on the target gene PRDM1 in esophageal carcinoma cell line EC9706

作　　者：李书军[1] 张利军 张海龙 牛秀兰 刘力娜[1] 魏素霞[1] 张合林[1] 

机构地区：[1]河北医科大学第二医院胸外科,石家庄050051 [2]河北省涉县中医院肿瘤科,邯郸056400

出　　处：《肿瘤》2011年第9期813-818,共6页Tumor

摘　　要：目的:构建微小RNA(microRNA,miR)-181a的真核表达载体和靶基因PRDM1(encoding PR domain containing1,with ZNF domain)的3'非翻译区(3'-untranslated region,3'UTR)荧光素酶报告载体,在人食管癌EC9706细胞中验证miR-181a对靶基因PRDM1的调控作用。方法:根据miR-181a成熟序列在基因组中的位置及其上下游200个碱基序列,以人食管癌KYSE150细胞基因组DNA为模板设计引物,PCR扩增包含miR-181a前体的序列,克隆到线性化的pMD18-T Simple载体中,经BamHⅠ和EcoRⅠ酶切后亚克隆到质粒pCDNA3.1(+)上,对重组质粒进行酶切鉴定和测序分析。通过实时定量PCR法检测人食管癌EC9706细胞转染重组表达载体pCDNA3.1-miR-181a后成熟miR-181a的表达水平。应用生物信息学预测软件对miR-181a的靶基因进行预测,将候选靶基因PRDM1的3'UTR融合到pMIR荧光素酶基因下游,通过双荧光素酶报告基因检测miR-181a对靶基因PRDM13'UTR的调控作用。将pCDNA3.1-miR-181a表达质粒转染到人食管癌EC9706细胞中,蛋白质印迹法检测其对PRDM1蛋白表达的影响。结果:成功构建miR-181a真核表达载体,实时定量PCR验证表明pCDNA3.1-miR-181a在EC9706细胞中能够过表达成熟miR-181a。生物信息学预测PRDM1可能是miR-181a的靶基因之一,于是构建了PRDM1的3'UTR荧光素酶报告载体pMIR-PRDM1,在此基础上对PRDM1的3'UTR"种子区"进行定点突变。双荧光素酶报告基因分析表明,miR-181a能够作用于PRDM1基因的3'UTR。蛋白质印迹法进一步证实,miR-181a能够抑制性调控内源性PRDM1蛋白的表达。结论:MiR-181a作用于PRDM1基因的3'UTR,在转录后水平上调PRDM1蛋白的表达,提示PRDM1是miR-181a直接调控的靶基因。Objective：To construct an eukaryotic expression vector of microRNA（miR）-181a and expression vector of 3’-untranslated region（3’UTR） of encoding PR domain containing 1 gene（PRDM1,with ZNF domain）,and to verify the regulation effect of miR-181a on the target gene PRDM1 in human esophageal carcinoma cell line EC9706.Methods：According to the genome position of mature miR-181a with its upstream and downstream 200 bp sequences,the genomic DNA of human esophageal carcinoma cell line KYSE150 was used as template to design the PCR primers,then the miR-181a precursor sequence was amplified by PCR.The PCR product was cloned into the linearized pMD18-T Simple vector and then subcloned into pCDNA3.1（＋） after double-ligation by BamHⅠ and EcoRⅠ.The recombinant plasmid pCDNA3.1-miR-181a was analyzed by restriction enzyme digestion and sequencing analysis.The expression level of mature miR-181a in human esophageal carcinoma cell line EC9706 transfected with pCDNA3.1-miR-181a was determined by using real-time PCR.The target genes of miR-181a were predicted by using bioinformatics software.The 3’UTR of candidate target gene PRDM1 was cloned into the downstream of luciferase gene in the pMIR-Report vector.The regulatory effect of miR-181a on 3’UTR of candidate target gene PRDM1 was determined by dual-luciferase reporter assay.Western blotting was used to detect the expression of PRDM1 protein in EC9706 cells transfected with pCDNA3.1（＋）-miR-181a.Results：The miR-181a recombinant eukaryotic expression vector was constructed successfully and it could effectively express mature miR-181a in EC9706 cells transfected with pCDNA3.1（＋）-miR-181a.PRDM1 was predicted as a candidate of target genes by bioinformatics.The point mutation analysis was performed in the ＂seed region＂ of PRDM1 3’UTR on the basis of construction of PRDM1 3’UTR luciferase reporter vector pMIR-PRDM1PRDM1.Dual-luciferase reporter assay showed that miR-181a could target the 3’UTR of PRDM1 gene,and the expression of e

关 键 词：食管肿瘤 微小RNA类 基因表达调控 miR-181a PRDM1基因 

分 类 号：R735.1[医药卫生—肿瘤]