The Biological Role And Mechanism Of PLA2G6 In The Occurrence Of Cutaneous Malignant Melanoma

Part Ⅰ:Expression of PLA2G6 in melanoma tissues and cell linesObjective:To verify the expression level of PLA2G6 in CMM tissues and cell lines(M14,SK-MEL-28,A375,A875).Methods:Oncomine database was used to investigate the mRNA expression of PLA2G6 in CMM tissues and benign nevi.The CCLE database was used to explore the mRNA level of PLA2G6 in different tumor cell lines.The expression of PLA2G6 in human MC and melanoma cell lines M14,SK-MEL-28,A375,and A875,were detected by RT-qPCR and Western blotting.The mRNA expression of PLA2G6 in CMM and paired para cancer tissues was detected by RT-qPCR.Immunohistochemical staining of CMM and nevus tissues were used to detect the location and expression of PLA2G6 protein.Results:By using the Oncomine and CCLE online database,immunohistochemistry,RT-qPCR,and Western blotting analysis,we revealed that PLA2G6 was markedly up-regulated in CMM tissues compared to nevus tissues,as well as remarkably increased in vitro in SK-MEL-28 and M14 melanoma cell lines compared to human melanocytes.In vivo,PLA2G6 was also elevated in nine melanoma tissues compared to adjacent tissues.Conclusion:The expression of PLA2G6 is increased in both CMM tissues and cell lines,suggesting that PLA2G6 may promote tumorigenesis in cutaneous melanoma.Part Ⅱ:Construction of PLA2G6 stable knockdown cell by RNAi strategy and verification of the biological function of PLA2G6-knockdown M14 and SK-MEL-28 cells in vitro and in vivo.Objective:To construct stable PLA2G6-knockdown M14 and SK-MEL-28 cell lines by RNAi strategy and observe its change on the malignant behavior of melanoma cells.Methods:PLA2G6-targeted shRNA sequences were designed and their Lentiviral vectors were constructed,identified,and transfected into M14 and SK-MEL-28 cells.The stable cell strain screened by puromycin with the highest knockdown efficiency was used to proceed with the following experiments.Cell proliferation was detected by CCK8 and colony formation assay in vitro and subcutaneous xenograft assay in nude mice in vivo.The effect of migration and invasion ability was investigated by scratch and Transwell assays.Flow cytometry was used to detect cell apoptosis.Results:The shRNA sequence with the highest silencing efficiency in M14 and SK-MEL-28 cells was screened by using RT-qPCR and Western blotting.The results of the CCK8 assay showed that the OD values of the PLA2G6 knockdown group at 48 h and 72 h time points were lower than those of the control groups.The results of the clone formation assay showed that the number of cloning of the PLA2G6 knockdown group was reduced in SK-MEL-28 and M14 cells,especially in M14 cells.In SK-MEL-28 and M14 cells,the apoptosis rate of the PLA2G6 knockdown group was higher than that of the control group.In BALB/C nude mice injected with M14 cells,tumor growth was slower in the PLA2G6-deficient group than in the control group.By the end of the experiment,the tumor weight significantly reduced in PLA2G6-deficient group.Immunohistochemistry showed that the expression of proliferation marker Ki-67 was down-regulated in PLA2G6 knockdown tumors.Conclusion:PLA2G6 knockdown can inhibit proliferation and metastasis and promote apoptosis of melanoma cells.In vivo,knockdown of PLA2G6 inhibited melanoma growth in mice.Part Ⅲ:Proteomics analysis of PLA2G6 knockdown M14 melanoma cellObjective:To analyze the differential expression proteins after PLA2G6 gene knockdown in M14 cells and predict and verify the mechanism of PLA2G6 in melanoma by GO and KEGG methods.Methods:The differential expression proteins(DEPs)between the PLA2G6 knockdown group and control group were identified by tandem mass spectrometry quantitative proteomics technique.Then the differential protein expression was then analyzed by GO,KEGG,and some were verified by Western blotting.Results:A total of 7231 proteins and 128 DEPs were obtained,respectively.Among the DEPs,90 DEPs were up-regulated and 38 DEPs were down-regulated.The results of GO enrichment analysis showed that proteins were categorized into three ontologies:Biological Process(BP),Molecular Function(MF),and Cellular Component(CC).The most enriched GO terms of BP,MF,and CC were annotated as immune response,peptidase regulator activity,and extracellular region.In KEGG pathway enrichment analysis,the ferroptosis pathway was the most relevant Biological Process to the tumor,and three genes were mapped to the ferroptosis pathway,which included transferrin(TF),ceruloplasmin(CP),and ferritin light chain(FTL).All these 3 genes were up-regulated in PLA2G6 knockdown cells.Western blotting analysis showed that the protein expression of CP and FTL were significantly up-regulated in PLA2G6 knockdown cells,which were in high accordance with proteomic assay results.However,there was no significant difference in TF protein expression.Moreover,we also detected the expression of the ferroptosis-related protein,including PTGS2,SLC7A11,and GPX4.The results showed that GPX4 expression was decreased,and PTGS2 expression was increased in M14 and SK-MEL-28 cells,but SLC7A11 was only reduced in M14 cells.Conclusion:The potential mechanism of PLA2G6 in CMM may be related to the ferroptosis pathway,and ferroptosis-related proteins are differentially expressed in PLA2G6 knockdown SK-MEL-28 and M14 cells.The biological function of PLA2G6 in melanoma and its association with ferroptosis may inspire PLA2G6-targeted therapeutic strategies.