The Molecular Mechanism Underlying Zinc Finger Protein PRDM5 In Promoting Cell Proliferation And Metastasis Of Melanoma

BackgroundMelanoma is the most dangerous skin cancer due to its highly metastatic potential.It is important to further elucidate the molecular mechanism underlying the metastasis of melanoma cells PRDM(PRDI-BF1 and RIZ domain containing)proteins constitute a family of zinc finger proteins and play important roles in multiple cellular processes by acting as epigenetic modifiers.Several studies suggest that PRDM5 is silenced due to the promoter hypermethylation in breast cancer,liver cancer,lung cancer,ovarian cancer,cervical cancer and gastrointestinal cancer.PRDM5 over-expression may induce cell cycle arrest at G2/M phase and apoptosis in ovarian cancer,breast cancer cells and hepatocellular carcinoma cells.However,the expression and role of PRDM5 in melanoma remain to be delineated.Objectives1.Explore the function of PRDM5 in melanoma cell proliferation,migration and invasion;2.Explore the function of PRDM5 in melanoma proliferation;Methods1.The function of PRDM5 in melanoma proliferation;PRDM5 overexpression and gene silencing plasmids were transfected into B16F10 cells respectively and cell proliferation was determined by MTT assay.Cells stably transfected with PRDM5 gene silencing plasmid was subcutaneously injected into mice,mice were sacrificed 2 weeks later and the tumor size was determined.2.The function of PRDM5 in melanoma migration and invasion.After transfection with PRDM5 overexpression or gene silencing plasmids,cells were cultured in serum-free medium for 24 h and a wound was generated by scratching themonolayer with a 200-μl sterile pipette tip.The cell-free area was photographed at different time points,and the wound healing was evaluated by calculating the percentage of cell-free area to the initial wounded area.After transfection,the migratory and invasive potential of melanoma cells was evaluated by Transwell assay with NIH-3T3 fibroblast conditioned medium as chemo-attractant.Cells bearing PRDM5 gene silencing or control plasmids were injected via the tail vein,and mice were euthanized 2 weeks later.Lung metastatic nodules were counted to determine the role of PRDM5 in the metastasis of melanoma in vivo.3.The molecular mechanism underlying PRDM5 inpromoting melanoma progression;Western blot was performed to examine the expression and activation of JNK and ERK signaling pathways.Results1.PRDM5 stimulates melanoma cell proliferationIndirectly,the number of living cells was detected by MTT method.PRDM5 gene overpression promotes proliferation.siCTRL and siPRDM5cells weresubcutaneouslytransplantedintomicerespectively.At week 2,the size and weight of melanoma in siPRDM5 cell-grafted mice was increased but still much smaller than those in siCTRL cell-grafted mice.2.PRDM5 promote melanoma cell migration and invasionThe wound inflicted on PRDM5 overpression cell monolayer was completely healed at the earliet by wound healing assay.PRDM5 silencing retards melanoma cell migration and invasion.3.PRDM5-promote melanoma progression is dependent on JNK and ERK signaling pathway.Western blotting indicated that the level of the phosphorylated JNK and ERK was increase,when the PRDM5 gene was overexpressed.However,the cells were treated with inhibitor,the phosphorylated JNK and ERK were decrease,the ability of melanoma cell migration and invasion was also inhbited.ConclusionsPRDM5 regulate melanoma progression through the JNK and ERK signaling pathways.