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

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.