The Role And Possible Mechanisms Of Exosome-derived ENO1 In The Growth And Metastasis Of Hepatocellular Carcinoma

【Background】Liver cancer is insidious and prone to recurrence and metastasis.At present,the clinical treatment of liver cancer is still limited.The latest global statistics show that liver cancer is the fourth most common cause of cancer-related death and is the second most lethal type of cancer,with a 5-year survival rate of 18%.According to histological classification,primary liver cancer can be divided into hepatocellular carcinoma,cholangiocarcinoma and combined hepatocholangiocarcinoma,of which hepatocellular carcinoma accounts for approximately 90% of primary liver cancer.Clinical data show that most hepatocellular carcinomas usually occurs in patients with hepatitis virus infection or alcohol abuse.In recent years,with the increase of patients with non-alcoholic fatty liver disease,metabolic syndrome and obesity,the risk of hepatocellular carcinoma also increases year by year.Liver cancer metastasis is a common clinical symptom and is also one of the major causes of death in patients with advanced liver cancer.Researches are different from traditional metastasis theory in recent years.Scientists generally believe that tumor metastasis can occur in the early stage or in the core area of tumors.During the evolution of tumors,tumor cells can first affect in situ microenvironment and the microenvironment of target organs by releasing extracellular vesicles,in order to form a kind of premetastatic niche that is favorable for tumor metastasis.Tumor metastasis is not a random process.Extracellular vesicles derived from tumor cells can activate a series of molecular programs,such as causing local immunosuppression and inflammatory response,enhancing angiogenesis,permeability and lymphangiogenesis,and prioritizing and reprogramming specific organs.Exosomes(50-150 nm in diameter),a class of extracellular vesicles,are nanosized vesicles surrounded by bimolecular lipid membranes and are formed by a series of precise regulatory processes,such as the "endocytosis-fusion-efflux" process.Exosomes usually contain a variety of biomolecules,including proteins,short-chain polypeptides,lipids,fragments of DNA,m RNA,micro RNA and so on.The components of exosomes are closely related to the cellular pathophysiological state,it mean that exosomes play an important role in the transduction of intercellular material and information.Notably,tumor cells can release significantly more exosomes than healthy cells,and the contents of tumor cell-derived exosomes differ from those of healthy cell-derived exosomes.This difference not only reflects the selective regulation of tumor-derived exosomes because of their structure and function but also suggests that tumor-derived exosomes facilitate tumor growth and metastasis through exosome–mediated crosstalk.As one of the important members in tumor microenvironment,multiple studies have shown that tumorderived exosomes participate in mulpitle biological processes,including intercellular signal transduction and tumor formation or deterioration.Proteins are the executors of physiological functions,their variability and diversity are involved in and influence the entire life activity process.A large of proteomics studies have confirmed that the protein expression profiles of hepatocyte and different liver cancer cells are significant different.The biological functions and related mechanisms of differential proteins have been elucidated one after another.The mapping of liver cancer protein profiles has become clearer.However,research on whether there are differences in protein expression in exosomes derived from different liver cancer cells,and whether related proteins can involved in regulating malignant progression of liver cancer is relatively insufficient.Therefore,based on the heterogeneity of metastatic potential of different HCC cells,this study aims to clarify the educational effect of high metastatic HCC cell-derived exosomes on low metastatic HCC cells.By comparing the protein expression profiles of exosomes derived from high metastatic HCC cells,low metastatic HCC cells and hepatocytes,we hope to further clarify the effects of high metastatic HCC cell-derived exosome-mediated proteins on HCC growth and metastasis,and explore their related molecular mechanisms that promote the malignant progression of HCC.The purpose is to provide new ideas for deep understanding of the progression of HCC to improve the clinical diagnosis and treatment for HCC.1.Select three types of HCC cell lines(MHCC97L,MHCC97 H,HCCLM3)withdifferent incidences of lung metastasis.Compare their proliferative activity,migratoryand invasive capacities,and activation of epithelial-mesenchymal transition via aseries of cell-based analyses in vitro.And distinguish between high and low metastaticHCC cells.2.Collect the exosomes derived from high metastatic HCC cells by ultracentrifugation.Identify exosomes by western blot,transmission electron microscopy and nanoparticletracking analysis.Use cell membrane staining kit(PKH67)to trace exosomes andobserve the uptake of exosomes derived from high metastatic HCC cells by lowmetastatic HCC cells.3.Detect the changes of proliferative activity,migratory and invasive capacities,andepithelial-mesenchymal transition of low metastatic HCC cells after high metastaticHCC cell-derived exosome education via CCK-8,Transwell,western blot and otherin vitro experiments.4.Use nude mouse model with orthotopic liver injection and tail vein injection to detectintrahepatic tumor formation and lung metastasis of low metastatic HCC cells afterhigh metastatic HCC cell-derived exosome education.5.Collect exosomes derived from hepatocytes and three types of HCC cells withdifferent metastasis potential to perform quantitative proteomics analysis of TMTmarkers.Analyze the differences in exosomal proteins expression and combine thedatabases to screen the key proteins in high metastatic HCC cell-derived exosomesthat may be involved in the malignant transformation of low metastatic HCC cells orpromote the malignant progression of HCC.6.To verify the hypothesis that the difference in exosomal ENO1 expression is due todifferences in its expression at cell level.Detect the expression of ENO1 in HCC cellsand tissues via western blot and immunohistochemistry staining,and analyze thecorrelation between ENO1 expression and clinicopathological characteristics of HCCpatients.7.Using lentiviral vectors to construct HCC cell lines with stable knockdown oroverexpression of ENO1.Collect exosomes derived from stable cell lines foridentification,labeling and tracing.Detect the expression level of ENO1 in exosomesderived from stable cell lines,and observe the effect of ENO1 expression in cells onthe secretion and uptake of exosomes.【Methods】8.After regulating ENO1 expression or educating with exosomes with high ENO1expression,detect the changes of proliferative activity,migratory and invasivecapacities,and epithelial-mesenchymal transition of HCC cells via CCK-8,Transwell,western blot and other in vitro experiments.9.After regulating ENO1 expression or educating with exosomes with high ENO1expression,use nude mouse model with tail vein injection to observe the occurrenceof lung metastasis of HCC cells.10.Search the literature,database and combine with bioinformatics analysis to predict thedownstream proteins and related signaling pathways regulated by ENO1.Use westernblot,immunofluorescence experiments to verify the influence of intracellular ENO1and exosome-derived ENO1 on downstream proteins and related signaling molecular.【Results】1.HCCLM3 cells have significantly higher proliferative activity,migratory and invasivecapacities,and significant activation of epithelial-mesenchymal transition comparedto MHCC97 L and MHCC97 H cells.2.High metastatic HCC cell(HCCLM3)-derived exosomes can enhance the proliferativeactivity,migratory and invasive capacities,and promote activation of epithelial-mesenchymal transition of low metastatic HCC cells(MHCC97L and Hep G2).3.High metastatic HCC cell(HCCLM3)-derived exosomes can promote intrahepatictumorigenesis and lung metastasis of low metastatic HCC cells(MHCC97L).4.Exosomes derived from hepatocytes(LO2)and HCC cells with different metastaticpotential(Hep G2,MHCC97 L,HCCLM3)have their own unique protein expressionprofiles.Exosomes derived from HCC cells partially reflect the characteristics of HCCcells.Some exosomal proteins are closely related to poor prognosis of HCC and areexpected to become potential biomarkers for HCC diagnosis and screening.5.Proteins involved in biological processes such as fluid shear stress,amino acidbiosynthesis,antigen processing and presentation,and cell carbon metabolism arehighly enriched in high metastatic HCC cell(HCCLM3)-derived exosomes.Theexpression of ENO1 showed an increasing trend with the enhancement of metastasisability in HCC cells.6.The exosomal ENO1 content arises from changes at cell level.High ENO1 expressionwas significantly associated with decreased overall survival.ENO1 expression wassignificantly associated with the tumor-node-metastasis stage and differentiationgrade.Univariate and multivariate Cox regression analyses identified ENO1expression as an independent predictor of the survival of HCC patients.7.Exosomal ENO1 expression was significantly reduced or increased in HCC cells withENO1 knockdown or overexpression.But,the regulation of ENO1 expression in cellsdo not affect the secretion or uptake of exosomes.8.High levels of ENO1 can enhance the proliferative activity,migratory and invasivecapacities,and promote activation of epithelial-mesenchymal transition in HCC cells.9.ENO1 knockdown can inhibite but ENO1 overexpression or exosome-derived ENO1can promote proliferation,migration and invasion,and the activation of epithelial-mesenchymal transition of HCC cells.10.Exosome-derived ENO1 can promote the lung metastasis and further growth of HCCcells.11.ENO1 knockdown can downregulate,while ENO1 overexpression can upregulate theexpression of integrin α6β4 in HCC cells.12.Exosome-derived ENO1 can affect integrin α6β4 expression and can activate integrin-mediated FAK/Src-p38 signal axis,thereby promoting the proliferation and metastasisof HCC cells.【Conclusion】In this study,based on the heterogeneous metastatic potential of different HCC cells,we clarified the educational effect of exosomes derived from highly metastatic HCC cells which can induce the malignant transformation of low metastatic HCC cells,enhance proliferative activity,migratory and invasive capacities,and promote activation of epithelial-mesenchymal transition of low metastatic HCC cells.And high metastatic HCC cell-derived exosomes promoted intrahepatic tumorigenesis and lung metastasis of low metastatic HCC cells.Quantitative proteomic analysis showed that the protein expression profiles of exosomes derived from hepatocytes and HCC cells with different metastatic potential were significantly different.Exosomes derived from HCC cells partially reflect the characteristics of HCC cells.ENO1 was frequently upregulated in HCC cells or tissues,with even higher expression in highly metastatic HCC cells or metastatic tissues as well as in exosomes derived from highly metastatic sources.ENO1 expression was closely related to the progression and deterioration of HCC.Mechanism studies showed that HCC cells with low ENO1 expression can upregulate integrin α6β4 expression after educating with exosomes of high ENO1 expression.Interestingly,exosome-derived ENO1 activates integrin-mediated FAK/Src-p38 signal axis,thereby promoting proliferation,migration and invasion,and inducing epithelial-mesenchymal transition,and eventually ledding to the growth and metastasis of HCC.This study confirmed that,during the evolution of HCC,high and low metastatic HCC cells generated in different directions of differentiation can promote the growth and metastasis of HCC through exosome-mediated interactions of key proteins.This provided new ideas for in-depth understanding of HCC progression to improve the clinical diagnosis and treatment for HCC.