Research On The Effect Of Silencing ATAD3A On Proliferation、Invasion And Migration Of Melanoma A375 Cells

background and objective: Melanoma is the skin cancer with highly metastatic rate and mortality.Surgical resection is an effective method for early melanoma treatment.However,the rapid proliferation and high invasion ability of melanoma cells make the treatment strategies including chemotherapy,radiotherapy,targeted therapy and immunotherapy appear to be not sufficient in the treatment of advanced melanoma.Therefore,it is of great significance to explore the pathogenesis of melanoma and to identify potential therapeutic targets for the prevention and treatment of malignant melanoma.Recent studies have found that ATAD3A(ATPase family AAA-domain containing protein 3 A),a member of the ATAD3 family,not only plays an important role in normal physiological process,but also abnormally expressed in breast cancer,glioma,prostate cancer and other tumors and can promote the tumor cell,s proliferation,invasion and migration,treatment resistance and other malignant biological behaviors.But the role of ATAD3 A in melanoma has not been reported.Based on this,this study aims to preliminarily explore the role of silencing ATAD3 A in proliferation、invasion and migration of melanoma cells.Methods: We performed lentivirus infection method,the silencing ATAD3 A lentivirus and control lentivirus were used to infect melanoma A375 cells and puromycin was used to screen and purify for obtain stable cell line,and the efficiency were verified by q RT-PCR(quantitative real-time PCR)and Western blot.The colony formation assay,CCK-8 assay,transwell invasion assay and wound healing assay were performed to explore the effect of silencing ATAD3 A on proliferation、invasion and migration of A375 cells,and Western blot were performed to further explore the expression of self-renewal associated proteins(NANOG,OCT4,SOX2)and invasion and migration related proteins(MMP2,SLUG,VIMENTIN)with silencing ATAD3 A in A375 cells.Results: the A375 cell with green fluorescent protein(GFP)after infected with silencing ATAD3 A lentivirus,and the expression of ATAD3 A in A375 cells after infected with silencing ATAD3 A lentivirus was decreased in both gene(sh Ctrl:1±0.245,sh ATAD3A:0.23±0.073,t=5.217,p<0.01)and protein levels by q RT-PCR and Western blot.The colony formation assay showed that the volume and efficiency(sh Ctrl:43.667±5.033,sh ATAD3A:22.667±2.517,t =6.464,p<0.05)of colony were decreased after knockdown of ATAD3 A in A375 cell.The CCK-8 assay showed that knockdown of ATAD3 A in A375 cell attenuated the proliferation ability(4days,sh Ctrl:1.226±0.069,sh ATAD3A:0.744±0.051,t=10.146,p<0.01),and Western blot showed that silencing ATAD3 A down-regulated the expression of self-renewal associated proteins including NANOG,OCT4 and SOX2.Moreover,transwell invasion assay showed that knockdown of ATAD3 A decreased the invasived cell on the lower chamber(sh Ctrl:535±93.407,sh ATAD3A:262.67±17.616,t=4.962,p<0.01),and wound healing assay also showed that silencing ATAD3 A slowed down the rate of wound healing(24H,sh Ctrl:88.304±4.567,sh ATAD3A:57.197±6.8058,t=6.574,p<0.01).Western blot showed that silencing ATAD3 A down-regulated the expression of invasion and migration associated proteins including MMP2,SLUG and VIMENTIN.Conclusion: silencing ATAD3 A can reduce the proliferation,invasion and migration abilities of melanoma A375 cells.