Study On The Mechanism Of ADAM9 Regulatory Protein In Hepatoma Cells Based On TMT Proteomics And Phosphorylated TMT Proteomics

Purpose: Hepatocellular Carcinoma(HCC)accounts for 90% of liver malignancies.A disintegrin and metalloproteinase(ADAMs)is a class of zinc-dependent transmembrane glycoproteins,among which ADAM9 is a member.The expression of ADAM9 is up-regulated in human liver cancer and can promote the occurrence and development of liver cancer.However,its specific mechanism of promoting the occurrence and development of HCC cells is not perfect.In this study,TMT markers combined with bioinformatics analysis technology were used to find the differential protein expression profiles of liver cancer cells with different expression levels of ADAM9,and the biological processes involved in the differential protein were screened out combined with bioinformatics technology to study the influence of ADAM9 expression level on the proteomics of the occurrence and development of liver cancer cells.Screening the downstream proteins regulated by ADAM9 is helpful to further clarify the molecular mechanism of the occurrence and development of HCC cells.Methods: The stable knockout and overexpression cell lines of ADAM9 were constructed by lentivirus packaging technique.The sh-RNA-ADAM9 knockout lentiviral vector and empty vector were transfected into HCC cell line MHCC97 H by cell transfection technique.Adam9 overexpressing lentiviral vector and empty vector were transfected into HCC cell line Huh-7.The stable transgenic strains were screened by purinomycin,and the effect of ADAM9 protein knockout and overexpression was verified by Western blot.The differentially expressed proteins in the effective knockout group(MHCC97H-sh RNA-ADAM9)and control group(MHCC97H-sh RNA-Ctrl)(A2 VS A1),ADAM9 overexpression group(Huh7-ADAM9)and control group(Huh7-Ctrl)and(B2 VS B1)were identified and screened by TMT quantitative proteomics and phosphorylation TMT quantitative proteomics,respectively.Target proteins were screened by GO enrichment analysis,domain analysis,KEGG pathway enrichment analysis,significant difference analysis,subcellular localization analysis,and phosphorylated upstream kinase prediction analysis.RESULTS: There were 278 differential proteins in TMT(Tandem Mass Tag)labeled effective knockout group(MHCC97H-sh RNA-ADAM9)and control group(MHCC97Hsh RNA-Ctrl)(A2 VS A1),of which 173 were up-regulated and 105 were down-regulated.For TMT labeled ADAM9 overexpression group(Huh7-ADAM9)and control group(Huh7-Ctrl)and(B2 VS B1),there were 250 differential proteins,of which 108 were up-regulated and 142 were down-regulated..A total of 838 phosphorylated peptides were differentially expressed in effective phosphorylated TMT knockout group(MHCC97H-sh RNA-ADAM9)and control group(MHCC97H-sh RNA-Ctrl)(A2 VS A1),of which 556 were up-regulated and 282 were down-regulated..The first five KEGG pathways with significantly enriched proteins of phosphorylopeptides differentially expressed were adhesion junction(upregulated),epithelial cell signal transduction(upregulated),ribosome(down-regulated),gap junction(up-regulated),and metabolic pathway(down-regulated).A total of 862 phosphorylated peptides were differentially expressed in effective phosphorylated TMT labeled ADAM9 overexpression group(Huh7-ADAM9)and control group(Huh7-Ctrl)and(B2 VS B1),of which 561 were upregulated and 301 were down-regulated.The top 10 KEGG pathways with significantly enriched phosphorylated peptides were adhesion plaque,gap junction,spliceosome,cell cycle,guidance of axons,synthesis,secretion and action of growth hormone,bactericidal action,MAPK signaling pathway,oxytocin signaling pathway,and DNA replication,which were all upregulated.The combined analysis showed that the differentially expressed proteins performed their functions mainly through phosphorylation of upstream kinases.In the prediction table of phosphorylated upstream kinases,54 phosphorylated upstream kinases were predicted in the A2 VS A1 group and 67 phosphorylated upstream kinases were predicted in the B2 VS B1 group.Based on the prediction of upstream kinases in the two groups,a differentially expressed protein MCM2(P49736)was screened out that was significant to the expression of ADAM9.In the A2 VS A1 group,MCM2 as a substrate was phosphorylated by ATR,but its phosphorylation status was unknown,and its phosphorylation status was upregulated by CDC7.In B2 VS B1 group,MCM2 as substrate was phosphorylated by CDK7.After CDC7 phosphorylation,the phosphorylation status of MCM2 was up-regulated.Conclusion: In the stable ADAM9 knockout group(A2 VS A1)and the overexpression group(B2 VS B1),the differentially expressed proteins were screened by TMT proteomics and TMT phosphoryl proteomics.The differentially expressed gene MCM2 related to the expression of ADAM9 was screened by comparing the differentially expressed proteins in the two groups,through upstream kinase forecast analysis,MCM2 in ADAM9 stable after knockout group is upstream kinase ATR phosphorylation,the phosphorylation state is unknown,MCM2 expression in ADAM9 group is upstream kinase CDK7 phosphorylation,the status of phosphorylation status to rise,It can be inferred that the overexpression of ADAM9 in HCC causes the phosphorylation of MCM2,which leads to a series of biological functions of HCC cells.