Abstract: Objective To construct the short hairpin RNA (shRNA)-expressing plasmid vectors specific for BRAF gene,and to test their effects in BRAF knockdown in human melanoma cell lines.Methods Two pairs of specific BRAF shRNA oligoes and a pair of randomly synthesized non-specific shRNA oligo were synthesized and inserted into plasmid pGenesil-1.Their fidelity was confirmed by double endonuclease digestion and sequencing.The constructed plasmids were transfected into human melanoma cell lines A375 and M14.The expression of BRAF mRNA and BRAF protein were detected by RT-PCR and Western blotting,respectively.Results The designed shRNA oligoes were precisely cloned into the plasmid pGenesil-1.The expression of BRAF mRNA and protein were down-regulated by specific plasmid braf 1 and braf 2,except to non-specific plasmid neg.The plasmid braf 1 was more effective,reducing BRAF gene expression by 90 per cent.Conclusions Plasmid mediated shRNA could successfully knockdown BRAF expression in human melanoma cells,and the suppression of the gene expression could maintain for 1 month at least.

Key words: Melanoma, RNA interference, BRAF, shRNA