Construction and in vitro identification of CXCR4 target plasmid vector of short hairpin RNA

Objective To construct a CXCR4 specific shRNA recombinant plasmid vector and identify its inhibiting effect on the gene expression of CXCR4 in melanoma cell lines.Methods A CXCR4 specific recombinant plasmid vector was prepared and transfected into the cultured MV3 cell line with lipofectamine 2000.RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4,respectively.Results CXCR4 specific shRNA was constructed and transfected into the MV3 cell line.Agarose gel electrophoresis confirmed the vector had been inserted in the production of PCR,and sequencing result showed that composite CXCR4-shRNA inserting correctly in the positive clone.Both the CXCR4 protein and mRNA expression in the CXCR4-shRNA group were significantly lower than that of the control group.Conclusion CXCR4-shRNA can be highly effectively transfected into melanoma cell,and induced post-transcriptional gene silencing of CXCR4,which may become a potential target in the prevention and treatment of invasion and metastasis of melanoma.