Role Of Cytoskeletal Protein Mena And Integrin ?9 In The Metastasis Of Hepatocellular Carcinoma

This dissertation contains two sections and focused on two genes,MENA and ITGA9,which have not yet been studied in hepatocellular carcinoma.We aim to investigate their influence on HCC metastasis and the underlying mechanism.The first section is about microfilament regulatory protein Mena in metastasis of hepatocellular carcinoma.Currently,most of the research on cytoskeletal protein in tumor development is about microtubules and their associated proteins,while research on microfilament system in oncology area is still in the early stage.We found MENA promoted metastasis of HCC cell in vitro and in vivo,and we made preliminary exploration on involved signal pathway.Firstly,we examined the expression of MENA in cells and tissues of hepatocellular carcinoma by Real-time PCR and HCC tissue microchip,and analyzed the association between Mena expression and metastasis risk according to the clinical indexes;We got MENA knockdown hepatocellular carcinoma cell lines by lentiviral infection,and carried out migration,invasion,anoiksis assays and immunofluorescence experiment with these cell lines,through which we observed on cellular level the effects of MENA on invasion and metastasis of hepatocellular carcinoma and its molecular mechanism;Furthermore,through Western Blotting,Pull Down experiment and gene chip detection,the changes on protein level,phosphorylated protein level and m RNA level in MENA knockdown HCC cell line were observed,and the signaling pathways affected by MENA were speculated;Finally,the influence of Mena on HCC cell metastasis in vivo was confirmed through nude mice tumor transplantation experiment.The results showed that,by HCC tissue microchip analysis,Mena expression showed to be positively associated with cancer thromb formation,tumor multiplicity,microvascular invasion and TNM staging;MENA downregulation inhibited migration and invasion of HCC cells,promoted its anoiksis,decreased cytoskeleton synthesis,and decreased focal adhesion and filopodia formation;As to the signaling pathway,Mena increased the STAT3,Erk and Rho A activity,while decreased the Cdc42 activity,which could partially explain the mechanism of MENA in promoting the survival,migration and invasion of HCC cell;Finally,both in nude mice liver orthotopic and vein injection transplantation tumor model,MENA showed to be a promotion factor on HCC cell metastasis in vivo.In summary,we found positive correlation between Mena expression and metastatic clinical index through HCC tissue microarray analysis.And the results show that MENA promoted HCC metastasis on the cellular,molecular and in vivo levels,which provides evidence for further exploration on the related mechanism of MENA and microfilament system in HCC metastasis.The second section mainly studies integrin ?9(ITGA9)in HCC development.Integrin mediates transduction between mechanical signal and molecular signal,and is closely related to adhesion and migration of tumor cells in tumor microenvironment.And it also plays an important role in regulation of the cytoskeleton.The research of ITGA9 will bring us to further understanding of the effect of cell mechanical structure,including cytoskeleton,on tumor metastasis.Through cellular level assays,we found that ITGA9 inhibited HCC cell proliferation and invasion,and promoted its anoikis.Based on these,we made subcutaneous,liver orthotopic and vein injection transplantation tumor model in nude mice.The result showed that ITGA9 inhibited HCC tumor formation,growth,in situ metastasis in liver and distant metastasis through blood vessel.On the other hand,we screened out Ran BP9 as a partner interacting with ITGA9 by yeast two-hybrid technique.The interaction between ITGA9 intracellular domain and Ran BP9 in human hepatic stellate cell LX-2 was verified by co-immunoprecipitation(Co-IP).Based on the preliminary study of ITGA9 in HCC shown above,ITGA9 was confirmed as a tumor suppressor in in vivo and in vitro experiments.And we also screened out and verify Ran BP9 as the protein partner interacting with ITGA9.These results provide foundation for further research on the molecular inhibition mechanism of ITGA9 in HCC development.