Study Of The Adjustment Mechanism For Merlin/NF2Protein To Liver Fibrosis In Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma (HCC), one of the most common fatal malignancies in China and many other countries in Asia and Africa, is the leading cause of cancer mortality in China.Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. It is a critical stage in cirrhosis development which the end stage consequence of fibrosis.Liver fibrosis, also Cirrhosis is very closely related to the occurrence of hepatocellular carcinoma. More than80percent of patients in China who develop liver cancer do so because they have cirrhosis of the liver.15%-25%, usually about20%cirrhosis patients develop liver cancer. The majority of liver cancer occurred on the base of posthepatitic cirrhosis.Chronic injury leading to fibrosis in liver occurs in response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drugs, metabolic diseases due to overload of iron or copper, autoimmune attack of hepatocytes or bile duct epithelium, or congenital abnormalities. The immune system is activated and the repair process swings into gear because of these insults. The injury or death (necrosis) of hepatocytes stimulates inflammatory immune cells to release cytokines, growth factors, and other chemicals. These chemical messengers direct support cells in the liver called hepatic stellate cells (HSCs)to activate and produce collagen, glycoproteins (such as fibronectin), proteoglycans, and other substances. These substances are deposited in the liver, causing the build-up of extracellular matrix (nonfunctional connective tissue). HSCs play a very important role in the process of liver fibrosis. Cytokines are also a critical factor in activated liver fibrosis. They interacted HSCs, ECM, and also interacted themselves, which constituted network-like control networks. TGF-β1is the most effective cytokine to improve hepatic fibrosis, which can inhibit the proliferation of liver cells and stimulate HSC activation, and promote the production of ECM and regulate the apoptosis of hepatocytes.The tumor suppressor NF2, which is inactivated in the familial cancer syndrome Neurofibromatosis type2, encodes for Merlin, a member of the Ezrin/Radixin/Moesin (ERM) family of proteins. Merlin and the closely related ERM proteins form a subgroup of the Protein4.1superfamily. NF2is a predominantly inherited disorder characterized by the development of schwann cell tumors and other brain tumors. Mutations or the loss of heterozygosity of the NF2locus has been detected in various tumors of the nervous system, such as schwannomas, meningiomas and ependymomas. Merlin is composed of an N-terminal FERM domain, an ensuing a-helical domain and a C-terminal domain that includes an actin-binding module in the ERM proteins but not in Merlin. The FERM domain adopts a cloverleaf structure composed of three interdependent lobes and seems designed to bring multiple proteins together at the membrane. This is well supported by the long list of proteins that have been reported to interact with the Merlin/ERM FERM domain, including transmembrane receptors such as CD43and CD44, and the tandem PDZ-domain-containing adapters Na+/H+exchanger regulatory factor (NHERF)-1and-2, which in turn associate with a variety of membrane receptors. The FERM domain can also associate with regulators of Rho GTPase signaling. The a-helical and C-terminal portions of Merlin can fold back and envelop the FERM domain, masking all known sites of protein interaction in both the FERM and the C-terminal domains, including the ERM actin-binding domain. For Merlin, evidence from studies of mammalian cells indicates that this self-associated’closed’form is the active growth-suppressing conforma-tion. In fact, many tumor-derived missense mutations are predicted to disrupt the closed conformation.Merlin is a membrane/cytoskeleton-associated protein that can link the membrane proteins to the underlying cortical cytoskeleton and in controlling the distribution of and signaling from membrane receptors. In mammalian cells, Merlin can block the internalization of ligand-bound EGFR specifically in contacting (confluent) cells in culture. In fact, whereas wildtype, Merlin-expressing cells normally downregulate EGFR signaling at high cell density, Merlin-deficient cells fail to do so and also fail to undergo contact-dependent inhibition of proliferation—a phenol-type reversed by pharmacologic inhibition of EGFR.Merlin and the incidence of HCC have a close relationship. In a recent study, the gene knockout mouse animal model, demonstrated Merlin/NF2protein is the critical inhibition in HCC. NF2homozygous mutant at the mouse leads to embryonic failure immediately before gastrulation, indicating that merlin function is critical at a very early stage in development. Nf2+/-mice developed a variety of malignant tumors later in life.63%developed osteosarcoma, followed by hepatocellular carcinoma (11%). Targeted deletion of NF2in the mouse liver results in massive hepatic enlargement due to OCs hyperproliferation and All mice eventually developed frank liver tumors then metastasized to lung. In adult mice which the liver specific NF2(-/-) gene knockout, liver OCs cell proliferation mildly, and basically does not occur HCC; but after stimulating the liver of these mice, such as partial hepatectomy (PHx), OCs cells was significant proliferation and induce HCC and CC. NF2(-/-) mice injected EGFR inhibitors Erlotinib, can inhibit the proliferation of OCs.Merlin was considered to be a strong disincentive in the development of liver cancer. Merlin for the occurrence of liver fibrosis research so far no one has. In China, hepatitis, cirrhosis of the liver cancer rate is high. Then Merlin can inhibit hepatic fibrosis? Merlin is the mechanism through which inhibition of liver fibrosis? Therefore, in-depth study to understand Merlin on liver cancer and liver fibrosis in relation to liver cancer, liver fibrosis development mechanism, as well as prevention and treatment of liver cancer, have put forward a new direction and new ideas.Method and Result:We used qPCR and Western blot detect the expression level of TGF-β1in four liver cancer cell lines:FOCUS、 MHCC-97H、HepG2、SMMC-7721and human normal liver cell line L-02. The results showed that:in FOCUS cells, the expression level of TGF-β1is the highest, and followed by the MHCC-97H, SMMC-7721, HepG2is the minimum; However, in normal human liver cell line L-02in both the mRNA or protein levels are difficult to detect the expression of TGF-β1. We detected by immunohistochemistry20cases of HCC specimens and10normal liver tissue levels of TGF-β1expression. TGF-β1in hepatocellular carcinoma strongly positive rate was55%(11cases), weak positive rate was40%(8cases), and negative in1case. TGF-β1expressed in the cytoplasm of cancer cells and stroma in hepatocellular carcinoma tissues. In all of the normal liver tissue, normal liver cells are not detected the expression of TGF-β1, while in liver endothelial cells can detect the strong TGF-β1expression. These results demonstrate that TGF-β1expression in HCC was significantly higher liver cancer cells themselves could secrete TGF-β1. Non-contact co-culture technique (Transwell chamber co-culture) was used to culture FOCUS cells and liver stellate cell line LX-2cells. Fluorescence quantitative PCR and Western blot confirmed that FOCUS hepatocellular carcinoma cells can secrete TGF-β1, activate TGF-β1/Smad pathway and promote synthesis of extracellular matrix (ECM) in LX-2cells.We used Western blot to detect the expression of Merlin and TGF-β1in four liver cancer cell lines:FOCUS, MHCC-97H, HepG2, SMMC-7721and human normal liver cell line L-02. The levels of expression of the two proteins are totally opposite. The cell lines with high expression of Merlin expressed low or absent TGF-β1, and Merlin missing cell lines expressed very high TGF-β1.In the same cell density, FOCUS cells were transfected with pCDNA-NF2plasmid and pCDNA3.1(+) plasmid. In the treatment group that transfected with pCDNA-NF2plasmid, Merlin gradually increased by improved the amount of plasmid, contrast to TGF-β1; but in the vector group, Merlin was always absent and TGF-β1was the same levels. It shows that Merlin controls the expression of TGF-β1, and recovery Merlin can reduce the expression of TGF-β1.In the same cell density, FOCUS cells were transfected with different concentrations of pCDNA-NF2plasmid and pCDNA3.1(+) plasmid. The FOCUS cells which were transfected with the plasmid and LX-2cells were co-cultured. We found that Merlin in FOCUS cells on the up room was gradually increased depend on the concentration of plasmid, and TGF-β1/Smad pathway of LX-2cells in the inferior well was gradual deactivation, such as p-Smad2, p-Smad3were declined; at the same time the synthesis of extracellular matrix (ECM) was reduced, such as the Fibronectin, Collagen Ⅰ, Collagen Ⅲ were also declined. This shows that resumption of Merlin in FOCUS cells can reduce the TGF-β1expression, promote to reduce the synthesis of ECM in hepatic stellate cell and inhibit liver fibrosis.The high expression of Merlin transfected with plasmid can significantly inhibit the epidermal growth factor receptor (EGFR) activation, thereby inhibit the expression of TGF-β1; and in the vector control under the EGF stimulation, the epidermal growth factor receptor (EGFR) is activated, TGF-β1expression along with the concentration of EGF was significantly increased in turn. The results confirmed that Merlin can inhibit the epidermal growth factor receptor signaling pathway, which reduced the secretion of TGF-β1.With the LX-2hepatic stellate cells gradually increased the number of cells, Merlin expression also gradually increased. In this linear cell density, in TGF-β1stimulation, the level of phosphated TGFBR Ⅰ receptor and Smad2in LX-2cells gradually reduced, TGF-β/Smad signaling pathway was inhibited. In the same conditions, due to Merlin in cancer cells FOCUS was loss, no matter how much the number of cells, FOCUS cells do not express Merlin. In the different cell density, stimulated by TGF-β1, TGF-β/Smad signaling pathway was activated continuously in FOCUS cells. In low cell density, hepatic stellate cell line LX-2cells expressed low Merlin, and high cell density with high expression of Merlin. In both cell density, stimulated by TGF-β1, low cell density LX-2cells compared with the high density TGF-β/Smad signaling pathway was significantly activated; its ability to greatly increase the synthesis of ECM. Meanwhile, in the low cell density, we transfected pCDNA-NF2to improve the expression level of Merlin, and then stimulated with TGF-β1and its TGF-β/Smad signaling pathway was significantly inhibited, ECM synthesis was significantly inhibited. On the other hand, in the high cell density, transfected shRNA to interfere Merlin, reducing the expression level of Merlin, and then stimulated with TGF-β1and the inhibitions of TGF-β/Smad signaling pathway and ECM synthesis were rescinded.Conclusion:In summary, this study was to detect TGF-β1protein levels in the HCC cell lines and hepatocellular carcinoma patients, and identified in hepatocellular carcinoma and liver cancer cell lines the TGF-β1protein expression were up-regulated; HCC cells can secrete TGF-β1themselves, and activated hepatic stellate cells TGF-β1/Smad pathway, promote hepatic stellate cells to synthesize extracellular matrix, and promote liver fibrosis. Merlin can inhibit the epidermal growth factor receptor signaling pathway, which reduced the expression of TGF-β1secretion. Restored Merlin in HCC cells can reduce the expression of TGF-β1, inhibit the hepatic stellate cell synthesize ECM, thereby inhibit the occurrence of liver fibrosis. Meanwhile, Merlin can negatively regulate TGF-β/Smad signaling pathway, and suppress liver fibrosis.