利用巢式PCR扩增目的基因构建hsa-miR-200c真核表达载体及鉴定  

Construction and identification of eukaryotic expression vector of human miR-200c by nested-PCR

作　　者：陈登宇[1] 陈峻崧[1] 王净[2] 曹文虎[1] 张洪义[1] 杨翠萍[1] 窦骏[1] 

机构地区：[1]东南大学医学院病原生物学与免疫学系,南京210009 [2]东南大学附属中大医院妇产科,南京210009

出　　处：《免疫学杂志》2012年第1期59-63,共5页Immunological Journal

基　　金：国家自然科学基金资助项目(81071769)

摘　　要：目的用巢式PCR从人类基因组获取miR-200c及侧翼序列,构建hsa-miR-200c重组体,转染卵巢癌细胞系SKOV3,检测其对E-钙粘蛋白表达的影响,为miR-200c对肿瘤细胞基因表达调控奠定基础。方法据microRNA数据库设计人miR-200c及侧翼引物,提取人细胞基因组进行PCR和巢式PCR获得miR-200c及侧翼共307个碱基序列,连接到pIRES2-EGFP载体中,测序鉴定后再将的miR-200c基因经慢病毒载体系统转染SKOV3细胞,检测miR-200c过表达对卵巢癌细胞E-钙粘蛋白表达的影响。结果成功扩增出miR-200c及侧翼共307个碱基序列,构建的重组体pIRES2-EGFP-miR-200c测序正确,转染SKOV3细胞后miR-200c过表达引起E-钙粘蛋白表达增多。结论巢式PCR技术能有效地从基因组中扩取miR-200c及侧翼片段用以构建miR-200c表达载体,miR-200c过表达可引起卵巢癌SKOV3细胞E-钙粘蛋白表达增多,可能导致细胞间黏附性增高。Research shows that hsa-miR-200c plays an important role in ovarian cancer metastasis.As hsa-miR-200c might be used to control ovarian cancer metastasis,we amplified hsa-miR-200c and its flanking sequence by nested-PCR to construct its eukaryotic expression vector,so as to study its regulatory effect on E-cadherin expression.According to hsa-miR-200c and its flanking sequence from miR data base,nested-PCR was performed to amplify expected product.The target gene was digested by double endonuclease,and cloned into the pIRES2-EGFP vector.The recombinant was identified by double endonuclease digestion and sequencing finally.Then the recombinant containing miR-200c gene was transfected into SKOV3 cells by lentivirus vector system.Forty-eight hours after the transfection,the miR-200c expression was detected by RT-PCR,and E-cadherin expression was detected by Western blot.We used nested-PCR assay to amplify miR-200c and its flanking sequence from human genome,and constructed miR-200c expression vector successfully.We found that more E-cadherin expression in SKOV3 cells with miR-200c transfection compared with the SKOV3 cells with mock transfection.It is an effective method to use nested-PCR to amplify microRNAs and flanking sequence from human genome,and the constructed miR-200c expressive vector is helpful to study its regulatory function on E-cadherin expression.

关 键 词：巢式PCR MIR-200C 卵巢癌 SKOV3细胞 

分 类 号：Q78[生物学—分子生物学]