关键词: shRNA;黑素瘤;BRAF基因;RNA干扰

Abstract:

Objective To investigate the effect of BRAFV600E mutation on the invasion capacity of a human melanoma cell line, A375. Methods Plasmids containing short hairpin RNAs （shRNA） specific for BRAF gene were prepared in previous study, and used to transfect A375 cells. Those cells transfected with negative plasmid and untransfected cells served as the controls. Transwell chambers were used to examine the invasion ability of melanoma cells in vitro. RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of matrix metalloproteinase 2 （MMP-2） and vascular endothelial growth factor （VEGF）, respectively, before and after the transfection. The activity of MMP-2 was also studied with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. Results Compared with the negative control, the specific shRNA decreased the mRNA and protein expressions of MMP-2 by 35% and 85%, respectively, and those of VEGF by 45% and 14%, respectively. Additionally, the number of cells invading through Matrigel chambers reduced by 69% in those cells transfected with the positive plasmid. Conclusions The mutant BRAFV600E has the potential to enhance the invasion capacity of melanoma cells, whereas specific shRNA could suppress the increase in metastasis capacity likely by inhibiting the production of VEGF and MMP.

Key words: shRNA;melanoma;BRAF;MMP-2;VEGF