Proteome And Ubiquitome Analysis Of Hepatocellular Carcinoma Cell Lines With Different Metastasis Ability

Hepatocellular Carcinoma(HCC)is one of the most common malignant tumor,which is causing the second leading cancer-related death worldwide.Despite remarkable advances in diagnostic and therapeutic techniques,the prognosis of HCC patients is still unsatisfactory.The 5 year recurrence/metastasis rate was even near 57.2%,which is the main bottleneck to improve the prospective outcome of HCC.Therefore,searching for novel prognostic biomarkers with high specificity and sensitivity in HCC is the key to improve the therapeutic outcomes,reduce mortality and increase long-term survival rate of HCC.By using label free based proteomics combind with liquid chromatography tandem mass spectrometry(LC-MS/MS),the present study aims to investigate the proteome and ubiquitome of hepatocellular carcinoma(HCC)cells with different metastasis ability(Hep3B,MHCC97L,MHCC97H and HCCLM3),to screen some differentially expressed proteins and ubiquitination site.We hope to find several potential prognostic biomarkers for predicting the recurrence/metastasis of HCC and discuss the molecular mechanisms of the metastasis of HCC.The proteins were considered to be differentially expressed if the iBAQ ratios were>2.0 or<0.5 when the highly metastatic HCC cell lines(MHCC97L,MHCC97H and HCCLM3)compared with the lowest metastatic HCC cell line(Hep3B).With the quantitative proteomics strategy,we identified 218 differentially expressed proteins in four HCC cell lines,of which 43 were up-regulated and 175 were down-regulated.The differentially expressed proteins were submitted to GO annotation and the results showed that these differentially expressed proteins were mainly involved in cell adhesion in biological processes and cadherin binding activity involved in cell-cell adhesion and Ran Guanosine triphosphatase activity in molecular functions.With KEGG signal pathway analysis,these differentially expressed proteins were mainly involved in carbon metabolism signaling pathway.And the protein-protein interaction networks of these differentially expressed proteins were mainly centered on HSPA9.Double glycine(di-Gly)antibody affinity purification was used to enrich the ubiquitinated peptides.In the quantitative proteomics,the ubiquitinated proteins and peptides were considered to be differentially expressed if the iBAQ ratios were>2.0 or<0.5 when the highly metastatic HCC cell lines(MHCC97L,MHCC97H and HCCLM3)compared with the lowest metastatic HCC cell line(Hep3B).In total,235 differentially expressed ubiquitinated proteins were identified,of which 70 were up-regulated and 165 were down-regulated.GO annotation revealed that these differentially expressed ubiquitinated proteins were mainly involved in cell adhesion and lactic acid metabolism in biological processes,and cadherin binding activity involved in cell-cell adhesion in molecular functions.With KEGG signal pathway analysis,these differentially expressed ubiquitinated proteins were mainly involved in glycolysis and glyconeogenesis signaling pathway.And the protein-protein interaction networks of these differentially expressed ubiquitinated proteins were mainly centered on CTNND1.Besides,the most probable motif is IALAEEAKAAGAPKE,and the main ubiquitinated sites were DEST K19;FLNB K1955.In conclusion,the present study investigated the proteome and ubiquitome of hepatocellular carcinoma(HCC)cells with different metastasis ability.This project would provide us better understanding of HCC recurrence and metastasis,and two potential novel targets,DEST and FLNB,for HCC prognosis and treatment.