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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 a 3’-untranslated region (3’UTR) luciferase gene encoding the eukaryotic expression vector of microRNA (miR) -181a and PRDM1 (encoding PR domain containing 1, with ZNF domain) Enzyme reporter vector to verify the regulatory effect of miR-181a on target gene PRDM1 in human esophageal carcinoma EC9706 cells. Methods: According to the position of the mature miR-181a gene in the genome and upstream and downstream of 200 base pairs, the primers of genomic DNA of human esophageal cancer KYSE150 cells were used as template to amplify the sequence containing miR-181a precursor and cloned into The linearized pMD18-T Simple vector was subcloned into plasmid pCDNA3.1 (+) after digestion with BamHⅠand EcoRⅠ, and the recombinant plasmid was identified by restriction analysis and sequencing analysis. Real-time quantitative PCR was used to detect the expression of mature miR-181a in human esophageal carcinoma EC9706 cells transfected with the recombinant expression vector pCDNA3.1-miR-181a. The bioinformatics prediction software was used to predict the target gene of miR-181a. The 3’UTR of the candidate target gene PRDM1 was fused to the downstream of the pMIR luciferase gene. The dual luciferase reporter gene was used to detect the effect of miR-181a on the target gene PRDM13 ’ UTR regulatory role. The pCDNA3.1-miR-181a expression plasmid was transfected into human esophageal carcinoma EC9706 cells. The expression of PRDM1 protein was detected by Western blotting. Results: The miR-181a eukaryotic expression vector was constructed successfully. Real-time quantitative PCR showed that pCDNA3.1-miR-181a overexpressed mature miR-181a in EC9706 cells. Bioinformatics Prediction PRDM1 may be one of the target genes of miR-181a. Then, a 3’UTR luciferase reporter vector pMIR-PRDM1 of PRDM1 was constructed, and then the 3’UTR “seed region” of PRDM1 Site-directed mutagenesis. Dual luciferase reporter gene analysis showed that miR-181a can act on the 3’UTR of the PRDM1 gene. Western blotting further confirmed that miR-181a inhibited the expression of endogenous PRDM1 protein. Conclusion: MiR-181a acts on the 3’UTR of PRDM1 gene and up-regulates the expression of PRDM1 protein at the post-transcriptional level, suggesting that PRDM1 is the direct target of miR-181a regulation.