The Involvement Of Neddylation In Melanoma Genesis

Melanoma is one of the most lethel malignancies and prone to be resistant to current therapeutic methods. The process of NEDD8covalent ligation to cullin proteins would promote the ubiquitination extremely that plays a critical role on the degradation of many proteins including cell cycle regulators. Previous research showed that NEDD8conjugation was up-regulated in the highly proliferative cell lines and was relevant to several tumors. Melanoma like other malignant tumors grow and metastasize rapidly. In this report we examined the NEDD8conjugataion and the expression of enzymes which modified the process of neddylation in three melanoma cells. To investigate the role of NEDD8conjugataion in melanoma genesis, we also studied whether inhibition of endogenous NEDD8conjugation had effects on the proliferation and invasion of melanoma cells by shRNA.Expression of NEDD8conjugation in tissues and cell linesTo confirm whether the level of NEDD8conjugation in melanoma cells were up-regulated, we applied western blot to analyse the amount of NEDD8conjugation in melanoma cells A375, M14, MV3, menalocytes and in seven pairs of melanoma, normal tissues around melanoma and three intracutaneous naevi. The results showed that NEDD8conjugation raised remarkably in A375, M14, and MV3compared with menalocytes. And the NEDD8conjugation in melanoma tissues were higher than it in normal tissues around melanoma correspondingly and in naevi.Expression of NEDD8conjugation relatived proteins in melanoma cell lines and menalocytesGenes expression of UCH-L3, NEDD8, UBC12, UBA3, APP-BP1in three melanoma cells and menalocytes were test by real-time PCR. The expression of UBA3, UBC12and APP-BP1in melanoma cells were all up-regulated compared with them in menalocytes. Respectively, NEDD8was up-regulated in MV3and A375, but in M14it was down-regulated compared with it in menalocytes. And in MV3and A375, the UCH-L3was up-regulated, but in M14, it was the same with that in menalocytes. Contrary to NEDD8and UCH-L3, the UBA3and UBC12expressed the highest in M14.Down-regulated expression of UBA3in M14cells resulted in reduction of NEDD8conjugationAfter transfected the M14with three shRNA-UBA3and shRNAT-U6.2, the optimum shRNA against UBA3was determined by immunocytochemistry and western blot. The score of positive percentage X staining strength in M14/shRNA-UBA3was lower than that in nontransfected M14and in M14/shRNAT-U6.2. The interference effect of this shRNA on NEDD8conjugation was verified by western blot formerly. The NEDD8conjugation in M14/shRNA-UBA3was less than it in nontransfected M14and M14/shRNAT-U6.2. The results showed a significantly statistical difference (P<0.05). The results above approved that after interfered, the NEDD8conjugation decressed in M14following with the expression of UBA3descenting.Inhibition of NEDD8conjugation could influence the malignant biologic behavior of M14in vitroTo observe the effect of inhibition of NEDD8conjugation on cells proliferation and invasion capacity, cells viabilily were measured by CCK8assay, tumors formation test were observed in nude mice and cells invasion capability were observed by transwell test. Transfection of shRNA-UBA3had significantly effect on the growth and metastasis of M14companied with the down-regulated NEDD8conjugation. The proliferative capacity of M14declined and the cells arrested at G0/G1phase increased obviously. The number of invasive cells in M14/shRNA-UBA3were lower than that in nontransfected M14and M14/shRNAT-U6.2(P<0.05). These results suggest that NEDD8conjugation is required for cell proliferation and metastasis. ConclusionsFor all the results above, we take the review that high expression of NEDD8conjugation might be involved in the malignant biologic behavior of melanoma. Expression of NEDD8conjugation in melanoma tissues and in melanoma cell lines are all upregulated. Interferance of neddylation passway in some extend could depress the proliferation and metastasis potential of melanoma.