| Objective:Hepatic stellate cells (HSCs) are the main ECM-producing cells during liver fibrosis.Hepatic stellate cells activate and transdifferentiate into myofibroblast are the dominantevents in liver fibrosis. Furthermore, activated HSCs stimulate the proliferation, growth andmigration of Hepatocarcinoma cells in vitro and in vivo, and Hepatocarcinoma cellsstimulate the growth, migration of human HSCs in turn. Hepatocarcinoma cells-HSCscross-talk in the liver drives progression in hepatocellular carcinoma. CCN1/Cyr61, animportant extracellular matrix protein, is a member of CCN family. CCN1plays complexand important roles in wound healing fibrosis through regulating fibroblast. In nonalcoholicfatty liver disease mice, CCN1is expressed in hepatocytes and involved in macrophageinfiltration hepatic proinflammatory response. In our former study, we demonstrated thatCCN1’s expression is significantly higher in benign hepatic cirrhosis tissue, cancer-adjacenthepatic cirrhosis tissue and overexpression of CCN1promotes the proliferation andmigration of HepG2cells. In addition, several integrin subunits are located at HSCsmembrane and mediate the proliferation, migration and fibrogenic activation of HSCs. Itmeans that CCN1may have potential roles in activation of HSCs. In this study, we plan todemonstrate the presumptions following: expression of CCN1level is elevated in liverfibrosis mouse model and is positively correlate with the progression of liver fibrosis,CCN1promotes the fibrogenic activation of HSCs through their direct binding to integrinαv subunit, and CCN1enhances the founction of HSCs in driving progression ofhepatocellular carcinoma in vitro and in vivo.Methods:1. The expression of CCN1in liver fibrosis and liver cirrhosis.1.1The expression and location of CCN1protein in human liver cirrhosis samples.1.1.1The expression of CCN1protein in32human liver cirrhosis samples,30 cirrhotic tissues adjacent to hepatocellular carcinoma and5normal human livers weresubiected to immunohistochemical staining.1.1.2The location of CCN1protein in human liver cirrhosis samples were determinedby immunofluorescent staining.1.2The expression and location of CCN1protein were dynamically observed duringthe progression of liver fibrosis mouse model.1.2.1During the progression of liver fibrosis mouse model by carbontetrachlorideinducing, the severity of liver fibrosis, the expression of collagenous fiber and expression ofCCN1protein in liver tissue were dynamically observed by HE staining, SIRIUS REDstaining, immunohistochemical staining and Western-blot, respectively.1.2.2Stop carbontetrachloride inducing liver fibrosis mouse model, the severity ofliver fibrosis and expression of CCN1protein in liver tissue were dynamically observed byHE staining, SIRIUS RED staining, immunohistochemical staining and Western-blot,respectively.1.2.3The location of CCN1protein in liver fibrosis mouse model were determined byimmunofluorescent staining.1.3The expression of CCN1protein was detected by Western-blot.2. The study of over-expressing the exogenous CCN1promoting the activation ofhepatic stellate cells and mechanism.2.1Recombinant adenovirus vector AdCCN1and AdRFP were amplification inHEK-293cells. Adenovirus titer were detected using HEK-293cells.2.2Hepatic stellate cells were infected with adenovirus recombinant adenovirus vectorAdCCN1or AdRFP, respectively. Fluorescence in hepatic stellate cells was observed usingfluorescence microscope. The proliferation of hepatic stellate cells and the expression ofCCN1, α-SMA and collagen Ⅰi nhepatic stellate cells were detected by MTT andWestern-blot, respectively.2.3The study of over-expressing the exogenous CCN1promoting the activation ofhepatic stellate cells through binding to integrin αv. After infecting with AdCCN1or AdRFPfor12hours, hepatic stellate cells were treated by function-blocking monoclonal antibodyagainst integrin αv. The proliferation of hepatic stellate cells and the expression of CCN1,α-SMA and collagenⅠ inhepatic stellate cells were detected by MTT and Western-blot, respectively.3. The study of CCN1enhancing the founction of hepatic stellate cells in drivingprogression of hepatocellular carcinoma.3.1The study of CCN1enhancing the founction of hepatic stellate cells in drivingproliferation of hepatoma carcinoma cells.3.1.1After infecting with AdCCN1or AdRFP for24hours, conditioned medium ofhepatic stellate cells was collected to treat HepG2cells. The proliferation of HepG2cellswas detected by MTT.3.1.2HepG2cells and different hepatic stellate cells were cocultured in6-well plateand transwell inserts. After five days, colony formation of HepG2cells was assaied byCrystal Violet staining.3.1.3After infecting with AdCCN1or AdRFP for24hours, conditioned medium ofhepatic stellate cells was collected to treat HepG2cells. The expression of tumor correlatedsignaling molecules in HepG2cells was detected by Western-blot.3.2The study of CCN1enhancing the founction of hepatic stellate cells in drivingmigration and invasion of hepatoma carcinoma cells.3.2.1After infecting with AdCCN1or AdRFP for24hours, conditioned medium ofhepatic stellate cells was collected to treat HepG2cells. The migration and invasion ofHepG2cells were detected by wound-healing-assay and transwell invasion assay.3.2.2After infecting with AdCCN1or AdRFP for24hours, conditioned medium ofhepatic stellate cells was collected to treat HepG2cells. The expression of invasioncorrelated signaling molecules in HepG2cells was detected by Western-blot.3.3The study of CCN1enhancing the founction of hepatic stellate cells in drivingprogression of hepatocellular carcinoma in vivo.3.3.1After LX-2cells were infected with AdCCN1or AdRFP for24hours, node micewere injected subcutaneously with HepG2cells alone, or LX-2cells alone, or HepG2cellsplus LX-2cells, HepG2cells plus LX-2-RFP cells, or HepG2cells plus LX-2-CCN1cells,respectively.3.3.2The length and width of tumor mass were dynamically measured. After beinginjected subcutaneously for twenty-six days, node mice were sacrificed. The tumors wereremoved and paraffin embedding. Total protein of the tumors were extracted. 3.3.3The morphous of subcutaneous tumor paraffin sections were observed by HEstaining.3.3.4The expression of collagenous fiber in subcutaneous tumor paraffin sections wereobserved by SIRIUS RED staining.3.3.5The expression of Ki67in subcutaneous tumor paraffin sections were assaied byimmunohistochemical staining.3.3.6The microvessel density in subcutaneous tumor tissues were observed byimmunohistochemical staining and immunofluorescent staining for CD31. The expression ofVEGF protein in subcutaneous tumor tissues were detected by Western-blot.Results:1. CCN1protein was expressed in liver cirrhosis and cirrhotic tissues adjacent tohepatocellular carcinoma, and was not expressed in normal human livers. The CCN1protein was mainly located in hepatocytes and was not expressed in activated humanhepatic stellate cells.2. During the progression of liver fibrosis mouse model, the expression tendency ofCCN1was closely associated with the severity of fibrosis: when liver fibrosis continuelyprogressed under CCL4injecting, the expression of CCN1was continuely heightened;when liver fibrosis relieved under no CCL4injecting, the expression of CCN1was reduced.CCN1was expressed in activated mouse hepatic stellate cells.3. CCN1was expressed at a low level in T6cells and not expressed in LX-2cells.4. Over-expressing CCN1promoted the proliferation of LX-2cells and T6cells, andstimulated the expression of collagen Ⅰand α-SMA.5. The founctions of CCN1promoting the proliferation and the expression of collagenⅠand α-SMA were inhibited when LX-2cells were treated by function-blockingmonoclonal antibody against integrin αv.6. CCN1enhanced the founction of LX-2cells in driving proliferation and colonyformation of HepG2cells, and the expression of tumor correlated signaling molecules inHepG2cells such as cycline D1、c-myc、survivin and CD34, and the activity of β-cateninsignaling.7. CCN1enhanced the function of LX-2cells in driving migration and invation ofHepG2cells, and the expression of invasion correlated signaling molecules in HepG2cells such as p-ERK1/2and MMP-9, and the inhibition of E-cadherin.8. The study of xenograft of nude mice found that LX-2cells alone did not result intumor formation, but all of the mice in other groups developed tumors at the site ofimplantation. The mice implanted with HSCs in addition to HepG2cells developed muchlarger tumors than the mice implanted with HepG2cells alone. Cotransplantation withLX-2-CCN1cells significantly promoted tumor growth compared with injection of LX-2orLX-2-RFP cells.9. HE staining of the subcutaneous tumor tissue revealed that the tissue structure ofHepG2cells plus LX-2-CCN1cells group was more complex than those of other groups. Thehyperplasia in multinuclear tumor cells, fibrous connective tissue, and infiltration ofinflammatory cells in HepG2cells plus LX-2-CCN1cells group were more obvious thanthose of other groups.10. According the result of SIRIUS RED staining, more collagenous fiber were foundin the subcutaneous tumors of HepG2cells plus LX-2-CCN1cells than those of other groups.Further, the positive staining of Ki-67was stronger in the subcutaneous tumors of HepG2cells plus LX-2-CCN1cells than those of other groups.11. The microvessel density and the expression of VEGF protein in the subcutaneoustumors of HepG2cells plus LX-2-CCN1cells were more than those of other groups.Conclusion:1. CCN1protein was expressed in liver cirrhosis and cirrhotic tissues adjacent tohepatocellular carcinoma. The CCN1protein was mainly located in hepatocytes. During theprogression of liver fibrosis mouse model, the expression tendency of CCN1was closelyassociated with the severity of fibrosis. It indicates that CCN1may involve in developmentof liver fibrosis-cirrhosis.CCN1was expressed in activated rat/mouse hepatic stellate cells and was notexpressed in activated human hepatic stellate cells. It indicates that CCN1may playdifferent roles in the progression of human and rat liver fibrosis.2. Over-expressing CCN1promoted the proliferation of LX-2cells and T6cells, andstimulated the expression of collagen Ⅰand α-SMA. Moreover, these founctions wereinhibited when LX-2cells were treated by function-blocking monoclonal antibody againstintegrin αv. It demonstrate that CCN1can promote the activation of hepatic stellate cells through binding integrin αv.3. CCN1enhanced the founction of LX-2cells in driving proliferation, migration andinvasion of HepG2cells. Furthermore, CCN1enhancing the founction of LX-2cells indriving the growth of HepG2xenograft in vivo. It demonstrate that CCN1enhancing thefounction of hepatic stellate cells in driving progression of hepatocellular carcinoma.Correlated mechanism may be that CCN1has effect on activated hepatic stellatecells-induced tumor microenvironment formation, which are beneficial for the progressionof hepatocellular carcinoma. |
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