Ubiquitinome Analysis And Potential Mechanism Of Hepatocellular Carcinoma Metastasis

Background Hepatocellular carcinoma(HCC)has a high incidence rate in China,and its risk factors include hepatitis B,hepatitis C and alcohol ingestion.Tumor metastasis is a major cause of death in many malignant tumors,including HCC,but its underlying mechanisms are still lacking in systematic research.Ubiquitination is an important type of posttranslational modification of proteins that has been shown to play a key role in the metastasis of a variety of tumors.Ubiquitination proteomics is a high-throughput method that combines immunoprecipitation with mass spectrometry to detect protein ubiquitination modification levels and sites.Therefore,the quantification of protein ubiquitination changes during HCC metastasis by ubiquitination proteomics is helpful for the study of the mechanism of HCC metastasis and the screening of potential drug therapeutic targets.Aims(1)To develop a high-throughput omics method that can quickly,efficiently and cheaply detect the ubiquitination levels of proteins in the human tumor.(2)The dynamic changes of general proteomics and ubiquitination proteomics during HCC metastasis were described,and the E3 ubiquitin ligase affecting HCC metastasis was screened and predicted.(3)To investigate the effect of E3 ubiquitin ligase SYVN1 on HCC growth and metastasis.(4)To explore the possible molecular mechanism of E3 ubiquitin ligase SYVN1 promoting HCC metastasis.Methods(1)By adjusting the experimental parameters of ubiquitination proteomics like protein desalination methods,trypsin digestion conditions,method of TMT markers,polypeptide fractionation methods,liquid fractionation method of MS,detection parameters of MS and the search library to maximize the detection number of ubiquitination site and minimize the cost of experiment under the condition of limited sample size.(2)15 HCC patients with vascular invasion and 15 HCC patients without vascular invasion were selected.The differences in protein expression levels between tumor tissues of 30 HCC patients and their corresponding peritumoral tissues were detected by general proteomics,and the differences in protein ubiquitination levels between tumor tissues of 30 HCC patients and their corresponding peritumoral tissues were detected by ubiquitination proteomics.The ubiquitination levels of protein in tumor tissues and peritumoral tissues were compared between HCC with vascular invasion and HCC without vascular invasion.Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligase associated with tumor metastasis.Finally,the topological network of protein substrate and corresponding E3 ubiquitin ligases was constructed and the key E3 ubiquitin ligases were found.(3)The growth,migration and invasion ability of LM3 and HUH7 hepatoma cell lines in the negative control group and the E3 Ubiquitin Ligase(SYVN1)expression interference group were compared by cell proliferation assay,subcutaneous tumor assay,pulmonary metastatic tumor assay,umphal vein endothelium tube formation assay,transwell migration assay and transwell invasion assay.(4)The interacting proteins of SYVN1 were screened and verified by protein interaction omics,immunofluorescence,and immunoprecipitation.Ubiquitin levels of related protein substrates in LM3 and HUH7 hepatoma cell lines were compared in negative control group,SYVN1 interference group and SYVN1 overexpression group.Results Using improved ubiquitination proteomics,11913 ubiquitination modification sites and 4317 ubiquitination modification proteins were identified in HCC.Among all the 11913 ubiquitination modification sites identified,2852 sites showed increased ubiquitination and 74 sites showed decreased ubiquitination in HCC.Among the 4317 identified ubiquitination modified proteins,1102 proteins showed increased ubiquitination and 17 proteins showed decreased ubiquitination in HCC.Most ubiquitination modification sites(6106)and ubiquitination modification proteins(3047)were identified between the two groups,but more ubiquitination modification sites(3694)and ubiquitination modification proteins(862)were identified in HCC with vascular invasion.In addition,HCC with vascular invasion has higher signal intensity and shorter ubiquitin chain length at the ubiquitination modification sites.Comparison of HCC with vascular invasion with HCC without vascular invasion showed that the ubiquitination modification sites with significantly increased signal intensity included K447 of EEF2 K,K64 of Smad5,and K254 of PLPBP.The ubiquitination modification sites with significantly reduced signal intensity included K1059 of SMC3,K64 of HMGN2 and K51 of CDA.The E3 ubiquitin ligase SYVN1 was found to be at the center of the E3 ubiquitin ligase topology by predicting the E3 ubiquitin ligase and constructing the network topology of proteins containing these ubiquitination modification sites.General proteomics data showed that the expression of SYVN1 in HCC with vascular invasion was significantly higher than that in peritumoral tissues,while there was no difference in the expression of SYVN1 in HCC without vascular invasion.Survival analysis showed that HCC patients with high expression of SYVN1 had a worse prognosis than HCC patients with low expression of SYVN1.Proliferation experiments in serum-free medium in vitro and subcutaneous tumor experiments in nude mice showed that the proliferation rate of LM3 and HUH7 hepatoma cells was positively correlated with the expression of SYVN1.Through transwell cell migration assay,we found that the migration ability of LM3 and HUH7 hepatoma cells with low expression of SYVN1 was weaker than that of LM3 and HUH7 HCC cells with normal expression of SYVN1.In addition,LM3 and HUH7 hepatoma cells with low expression of SYVN1 had weaker ability to promote the tube formation of HUVEC cell and lower expression levels of angiogenesis related genes such as b FGF than those in normal control group.Experiment of pulmonary metastases by caudal vein injection showed the metastatic ability of HCC cells in the SYVN1 stable knockdown group was worse than that in the normal control group.In addition,the transwell invasion assay also found that hepatoma cells in the SYVN1 stable knockdown group were less able to penetrate the matrix gel than those in the normal control group.Protein interaction analysis showed that HSP90AA1,HSP90AB1 and HSP90B1 of the heat shock protein family had high scores in LM3 and HUH7 cells.In addition,through the analysis of the general proteomic data,we also found that the expression level of SYVN1 in HCC was highly positively correlated with the expression levels of HSP90AA1,HSP90AB1 and HSP90B1.Immunoprecipitation and immunofluorescence experiments confirmed that both endogenous and exogenous expressions of SYVN1 interacted with HSP90.However,ubiquitination experiments in both hepatoma cell lines and 293 T cell lines showed that changes in SYVN1 expression did not affect the ubiquitination of HSP90.Ubiquitin proteomics data showed that the ubiquitination level of EEF2 K in HCC with vascular invasion was significantly higher than that in HCC without vascular invasion.Western blotting assay and transcriptome analysis of GEPIA database showed that EEF2 K was highly expressed in HCC.In addition,the E3 ubiquitin ligase of EEF2 K was predicted by Ubi Browser database to be SYVN1.Finally,we found that the ubiquitination level of EEF2 K decreased after the expression of SYVN1 was inhibited in HCC cells.Conclusion The improved ubiquitination proteomics was able to quickly and easily detect more than 10,000 protein ubiquitination modification sites from HCC samples.HCC tissues had higher ubiquitination modification level than peritumoral tissues,and HCC with vascular invasion had more ubiquitination modification sites and shorter ubiquitin chain length than HCC without vascular invasion.The ubiquitination modification levels of EEF2 K,TXNRD1,RASSF4 and other proteins were significantly increased in HCC with vascular invasion,and it was predicted that SYVN1 might be an E3 ubiquitination ligase regulating the ubiquitination levels of these proteins.SYVN1 promoted the proliferation of both hepatoma cell lines in vitro and in vivo.SYVN1 promoted angiogenesis and enhanced the migration and invasion of tumor cells in both hepatoma cell lines.SYVN1 interacts with HSP90 but does not affect the ubiquitination of HSP90.SYVN1 can promote the ubiquitination of EEF2 K.