The Expression Of Epm And Its Relation To Progression Of Hepatocellular Carcinoma In HCC Microenvironment

Background and aimsHepatocellular carcinoma (HCC) is one of the most common cancers in the world, especially in Asia and Africa, with a very poor prognosis. Most cases of HCC are secondary to either a viral infection (hepatitis B or C) or cirrhosis (alcoholism being the most common cause of hepatic cirrhosis). It is considered that the pathogenesis of HCC is a long-term process that involves multiple genetic or epigenetic alterations. For years researcher have focused on cancer cells itself only; recently we have begun to think more molecularly how the tumor microenvironment(the normal cells and moleculars that surround a tumor cell) influences the cancer cells. Tumor microenvironment is a complex system, which contains fibroblasts, infiltrating immune cells, extracellular matrix and cytokines. The communication between the cancer cells and the surrounds helps drive the process of the tumor progression. Some of the key steps of cancer progression depend on the surrounding microenvironment, for example, proliferation, metastasi, invasion and angiogenesis. However, most of the mechanisms are not well explored.Hepatic stellate cell, first described by Kuffer in 19th century, is remarkably versatile mesenchymal cells which is vital to hepatocellular function and the liver's response to injury. In 1994, Enzan discovered that hepatocellular carcinoma cells were always surrounded by actived hepatic stellate cells that were strongly positive for alpha-smooth muscle actin. After that, hepatic stellate cells, as an important member of liver cancer microenvironment, were brought more and more attention. Previous studies have suggested that stroma cells function as enhancers or suppressors through a paracrine fashion. They promotes HCC proliferation,metastasis and invasion in vitro.Epimorphin, also called syntaxin-2 and discovered by Hirai in 1992, is a multifunctional membrane protein with dual topology. Its C terminus is membrane anchored. When the N terminus is on the cytosolic face it acts as a t-SNARE involved in intracellular vesicle docking and when flipped inside out, i.e. N terminus hangs out on the extracellular surface (by some nonclassical secretion pathway) it acts as a versatile morphogen and is called epimorphin. When expressed by mesenchymal cells it can instruct epithelial morphogenesis at epithelial mesenchymal interfaces. The normal development (morphogenesis) and maintenance of many organs and tissues, including intestine, skin, mammary gland, lung, gallbladder, and liver, are dependent on interaction between mesenchymal and epithelial cells. Epimorphin has been identified as a mesenchymal molecule with recognized morphogenic effects.Deborah Rubin and colleagues examined epimorphin-null mice and show that epimorphin is required for the regulation of epithelia in many tissues, including during testicular development and normal spermatogenesis. In the gut, epimorphin was found to act as a negative regulator of gut epithelial cell proliferation, and epimorphin deficiency provided protection in an experimental model of colitis. These data suggest that modulation of epimorphin expression could be used therapeutically to increase mucosal regeneration following injury to the gut in inflammatory bowel disease, ischemia, or following surgical intestinal resection. In another study, epimorphin deletion inhibits chronic inflammation-associated colon carcinogenesis in mice, likely as a result of increased epithelial repair, decreased myofibroblast IL-6 secretion, and diminished IL-6-induced inflammation.Thus, EPM in the HCC may be involved in tumor angiogenesis and pathogenesis of hepatic carcinogenesis. The purpose of this study was to examine the involvement of EPM in the progression of hepatoma.Methods1. We investigated the effect of human HSC conditioned medium (CM) on the HCC cell proliferation.2. Scratch assay and matrigel invasion assay were used to analyse the cell invasion in HCCs that was cultured with HSC CM. Tumorigenicity and metastasis assays in nude mice were used to authenticate it in vivo.3. Preliminary analysis of the mechanism: we investigated the effect of HCC CM on HSCs messenger RNA or protein expression of various factors such as TGF,SDF-1,HGF,CK-8,CCL-5,EPM in HSCs;we also constructed five RNA interference vectors of EPM, got hepatic stellate cells whose EPM is stable interfered,estimate the influence of EPM in HCC proliferation,invasion and migration; additionally, 3D coculture system was also used to compare the ability of sphere4. Preliminary study the mechanism of EPM promotion in cell invasion: Using MMP-9 in the interference vector,we interfered MMP-9 gene expression in EPM stable lines,compared the invasion ability of hepatocellular carcinoma cells before and after interference5. Preliminary analysis the mechanism of upregulated EPM in HSC: Bioinformatics analysis was used to search the CpG islands in promoter region,we analysed the relationship of methylation status and gene expression to explore the possible mechanism of EPM increased. Results1. Hepatic stellate cells promoted HCC cell proliferation, migration and invasion. The results of proliferation analysis demonstrated that HSCs-conditioned medium promotes HCC growth in vitro. We used scratch assay and Matrigel assays to investigate the effect of HSCs on the carcinoma cell migration and invasion. The results showed that, in comparison with the control medium, HSCs-conditioned medium enhanced migration and invasion potential of HepG2, MHCC97H and MHCC97L cells significantly. To confirm the effect of HSCs on HCC proliferation, we mixed MHCC97H and LX-2 cells and injected them subcutaneously into the nude mice. The growth kinetics of the tumors containing HSCs were compared to those for the tumors formed by HCCs injected on their own. After four weeks, we found that the presence of LX-2 cells accelerated the growth of HCC97H tumors significantly.2. EPM was up-regulated in carcinoma cell-activated HSCs. To understand better how hepatic stellate cells affect cancer cell proliferation and invasion, we co-cultured them with HCC cancer cells and measured their expression levels for various molecules.We found that several genes, including HGF, CCL5, CK18, OPN, EPM, SDF-1, TGF-β, were up-regulated in HSCs after co-culture with HCC cancer cells.Using realtime-PCR and Western blot assay, we confirmed that EPM accumulated to a high level after co-culture with HCC cancer cells. EPM expression was analyzed further by Immunofluoresence, we found that in normal liver tissue EPM was present mainly along the sinusoidal lining of hepatocytes (position of quiescent HSCs) and around blood vessels. EPM expression was much higher in the stroma tissue surrounding tumors than in normal liver parenchyma tissues.Western blot analysis also confirmed that EPM expression was markedly higher in the tissues adjacent to the tumors than in the matched normal tissues. These results show that EPM is up-regulated in activated hepatic stellate cells in hepatocellular tumor stroma.3. Aberrant methylation in the gene of EPM (STX2) is associated with its overexpression. To determine whether the up-regulation of EPM was associated with aberrant methylation, the HSC cell line LX-2 was treated with 5-Aza, a DNA methyltransferase inhibitor. After 5-Aza treatment, EPM expression levels were dramatically increased, indicating that aberrant methylation of STX2 was associated with the up-regulation of EPM in HSC treated with HCC CM.4. EPM promoted HCC cell invasion and metastasis. To investigate the role of EPM secreted by HSCs on cancer cell invasion, we used two different siRNAs to inhibit EPM expression in LX-2 cells and then performed Matrigel invasion assays. The result suggested that knockdown of EPM expression in HSCs significantly reduces their effect on cancer cell invasion. 5. EPM promoted HCC cell invasion and metastasis via MMP9 pathway. To address if EPM promote cell invasion through MMP-9 expression, siRNA was used to knock down MMP-9 expression in EPM-expressing MHCC97H and MHCC97L cancer cells. We found that knockdown of MMP-9 was sufficient to abolish the increase in cell invasion, suggesting that EPM facilitated the process by up-regulating MMP-9.ConclusionHepatic stellate cells promoted HCC cell proliferation, migration and invasion. EPM was up-regulated in carcinoma cell-activated HSCs, the phenomenon was associated with aberrant methylation of EPM CpG island.HSCs promote hepatocellular carcinoma invasion and metastasis through EPM-MMP9.