The Role Of The Fgl2Prothrombinase In Xenografts HCCLM6Tumor And Chronic Hepatitis B

[BACKGROUND&OBJECTIVE]Viral hepatitis B (CHB) caused by the hepatitis B virus (HBV) is common and serious issue in China as well as world wide. There were about350million people infected with HBV globally. There are about10%of adults while90%of children become chronic HBV carriers after HBV infection. Among those about100-200million annuealy died due to HBV related end stage liver dieases including liver failure, cirrhosis and hepatocellular carcinoma.Fibrinogen-like protein2(fgl2)/fibroleukin belongs to fibrinogen-related protein superfamiiy. There are two forms of the proteins. One is type â…¡ transmembrane protein expressed on macrophages and endothelial cells, while another is soluble protein secreted by CD4+and CD8+T cells. The membrane fg12protein called fg12prothrombinase has serine protease activity, which can directly catalyze prothrombinase into activated thrombinase, initiating cascade coagulating reaction. In our previous study, increased fg12expression was detected in many tumor tissues, which were located mainly in the tumor cells, interstitial infiltrated cells and vascular endothelial cells. The fibrocectin deposition can be seen in the close area to the fg12positive cells. The inflammatory factors of tumor microenvironment included TNF-alpha, VEGF can induce human hepatocellular carcinoma cell lines of HCCLM6and human tumor infiltrating monocyte-macrophage cell line THP-1to a high expression of fg12. The highly expression of fg12activated the MAPK pathway directly or indirectly to promote liver cancer grafted tumor growth, invasion and angiogenesis, and enhance the tolerance of liver tumor cell apoptosis induced by TNF-a. The soluble fg12(sfg12) secreted by Foxp3+regulatory T cells has immune regulatory function. Sfgl2inhibits the proliferation of T cells and mature of bone marrow dendritic cells via fgl2-FcyRIIB receptor pathway. Sfgl2has also been shown to contribute the regulatory T cell function and suppression of autoimmune disease development in mouse model of autoimmune disease. In the process of development of viral hepatitis, T cells, NK cells as well as Treg cells in liver were found to have different functions. Treg cells could reduce the immune response of and reduce immune pathological damage of the liver coused by MHV-3virus infection, and promote the chronicity of the MHV-3induced hepatitis. Hepatic NK cells were increased rapidly and actived to secrete IFN-gamma and of TNF-alpha in Balb/cJ mouse post MHV-3infection. The hepatic injury of NK cells was demonstrated to be mediated via the Fas-FasL and NKG2D-NKG2DL pathways.In the present study, we aimed to further investigate the role of fg12in tumor growth and angiogenesis and related mechanisms. First of all we studied that whether the fg12was highly expressed in human hepatocellular carcinoma and the co-localization of its expression with fibrin deposition. Secondly we examined the correlation of fg12expression and the expression of thrombin receptor protein kinase receptor (protease-activated receptors, PARs). Further more, we investigated the molecular events of fg12expression, downstream PARs mediated activation of MAPK, and their pathological effects on tumor cell growth, tumor invasion and angiogenesis.Chronic hepatitis B has been recognized closely related with the devolepment of HCC. Sfg12was reported to be involved in choronic hepatitis C virus infection. Whether it plays a role in CHB worths investigation.The purposes of this study are as the followings:1. To investigate the contribution of fg12to xenografts HCCLM6tumor growth and angiogenesis2. To investigate the contribution of sfg12expression in CHB. Collect PBMC from20cases of chronic viral hepatitis and detect the fg12expression, then re-mixed Treg and PBMC culture and detect the function of CD8+T cells. [METHODS]1. The fg12/prothrombinase contributes to xenografts HCCLM6tumor growth and angiogenesis1) Fifteen patients (12males and3females, average age of52.4years old) with primary hepatic carcinoma were recruited at Tongji Hospital from2009-2011and their tumor tissues were collected for fg12expression by immunohistochemical staining.2) Transfection of fgl2-miRNA to establish stable fg12low expression HCCLM6cell line. Nucleofection of fg12expressed cDNA plasmid to establish high expression fgl2HCCLM6. And measurement of the fg12expression in these cell lines for confirmation.3) The involvement of protease-activated receptors (PARs) activation.Detection of PARs expression in HCCLM6and its correlation with fg12at mRNA via RT-PCR and protein via flow cytometry and confocal.4) Mearment of MAPK pathway activation and JNK, ERK, p38pathways down stream of PARs under fg12expression in HCCLM6via flow cytometry liquid chip (CBA) technology.2. the role of sfg2in chronic hepatitis B1) Detecting the sfg12expresson in PBMC of CHB patients via flow cytometry and confocal microscope.2) Separating CD4+CD25+CD127dim/-Treg cells from PBMC via magnetic beads, and cultured with CD8+T cells (PBMCs) the proliferation index of CD8+T cells was detected by flow cytometry. And the secretion of IFN-y in the mixed culture was mesaured by enzyme-linked immunosorbent assay (ELISA)[RESULTS]1. The fg12/prothrombinase contributes to xenografts HCCLM6tumor growth and angiogenesis1) fg12was highly expressed in hepatocellular carcinoma, and correlated with fibrin deposition;2) successful establishment of fg12high expression and knockdown HCCLM6cell lines model; 3) increased fg12expression up-regulated the PAR1and PAR3expression while had no effect on PAR2and PAR4expressions;4) endogenous fg12expression actived the MAPK pathways and these effects involved ERK, JNK and p38;5) blockage of PAR1and PAR3reduced the phosphorylation of ERK, p38, but not the phosphorylation of JNK, while blocking PAR2down-regulated JNK phosphorylation.2. The PBMCs from CHB patients highly expressed both soluble and type â…¡ membrane fgl2; sfg12secreted by Treg cells inhibited the proliferation of CD8+T cells.[CONCLUSION]1. Fg12contributes to HCC tumor growth and angiogenesis and this effect involved PARs and MAPK pathways. Downregulation of its expression might be of therapeutic significance in HCC.2. Sfgl2protein secreted by Tregs suppresses the proliferation and IFN-r sereation of CD8+T cells in CHB.