Effects Of Mutated BRAF～(V600E) Gene On The Growth And Invasion Ability Of Melanoma In Vivo And In Vitro

Cutaneous malignant melanoma (CMM) is one of the most malignant tumors. It is easy for invasion and metastasis. The incidence of CMM is rising rapidly. Recently, someatic mutation in BRAF gene was found in a majority of human melanomas, which is the most frequently mutated gene in human melanoma. The purpose of the study is to investigate the role of the BRAF mutation in the development, invasion and metastasis of malignant melanoma by using plasmid mediated RNA interference.Firstly, two pairs of shRNA oligos (Braf1 and Braf2) specific against human BRAF coding sequences were designed. One of them, Brafl, was directed to the most common mutation, BRAFv6^V600E in melanoma. Meantime, a pair of non-specific, randomly synthesized shRNA oligos (Neg) also was designed. Then, three shRNA-expressing plasmids (braf1, braf2 and neg) were constructed. By using Fugene-6 transfectin, these plasmids were transfected into human malignant melanoma cell line, A375. Cells in which the plasmid was transfected were selected in media containing antibiotic G418. The effects of BRAF silenced in vitro were confirmed by semi-quantity RT-PCR and Western blotting, respectively. Two plasmids which have varying degrees of inhibiting effect of BRAF mRNA and protein expression were found. One of the plasmids, braf1, has more effective than braf2. The protein expression level of BRAF was decreased by 90%.Secondly, the xenograft models were used to examine the efficacy of inhibiting mutant BRAF^V600E gene by RNAi in vivo. In one trial, the nude mice were injected into the right flank subcutaneous with the same amount A375 shRNA-containing cell or control cell. 24 days after the injection, tumors of braf1 group showed significantly smaller and lighter than those of neg group and control cell group. In another animal experiment, after xenograft models were established, the plasmids and transfectin were mixed. Then the mixed were injected into mice tumors three times with one day interval between injections. During the treatment and five days after injections, tumors of braf1 group growed slowly compared to other groups, the inhibition rate is 62%. Thirdly, the effects of BRAF silenced in vivo were also confirmed by semi-quantitative RT-PCR and Western blotting, respectively. The results showed that braf1 inhibited the expression of BRAF mRNA and protein, the latter was declined by 62%.Fourthly, before and after treatment, semi-quantitative RT-PCR and Western blotting were be used to detect the expression of MMP-2 and VEGF of tumor cells in vivo and in vitro. The activity of MMP-2 also was studied by enzyme method. The results showed that MMP-2 and VEGF expression in vitro and in vivo are different degrees of reduction after transfection. The reduction was more obvious in vitro than in vivo.Finally, the Transwell chambers and nude mice were used as the models of tumor invasion in vitro and in vivo, respectively. The results showed that the number of cells transduced with brafl plasmid through metrigel was reduced. The numbers of lung metastatic tumors with braf1 plasmid were also reduced in vivo.Above all, these results show that BRAF mutant gene can promote human melanoma cell growth and proliferation, enhance tumor cell invasion and angiogenesis capacity and improve their ability to transfer by produce more VEGF and MMPs, suggesting BRAF mutation has an important role in melanoma growth, invasion and metastasis, and may be a target for gene therapy in future.