A Study On The Roles And Mechanisms Of TGF-?1 Inducing HSC To CAF In Metastasis Of Hepatocellular Carcinoma

OBJECTIVE Hepatocellular carcinoma(HCC)is an aggressive malignancy worldwide.The survival rate of patients with HCC is predominantly influenced by the invasive and metastatic ability,in which cancer-associated fibroblasts(CAFs)plays an important role.Hepatic stellate cells(HSCs)are one kind of main liver mesenchymal cells.HSCs are closely associated with the invasion and metastasis of HCC.Based on the above and our previous studies,we hypothesize TGF-?1 could induce the transformation of HSC into CAF mediated in the microenvironment of HCC,thus accelerating hepatocellular carcinoma invasion and metastasis.Our research aims to verify that TGF-?1 will help HSCs transformed into CAFs in the microenvironment of HCC,which further enhances the invasive and metastatic capacity of hepatoma cells.Our results can help us to understand the effect of mesenchymal cells on invasion and metastasis of HCC.What's more,they may help to provide new strategies for HCC prevention and treatment via microenvironmental approaches.METHODThe tissue specimens and follow-up data of patients with liver cancer were collected,and the distribution and density of HSC and CAF in cancer tissue and normal tissue were observed clinicopathologically.The expression levels of TGF-?1 and CAF marker molecules(?-SMA,FAP)were detected by immunohistochemistry,and the correlation between the distribution and density of TGF-?1 and HSC,CAF and the invasion and metastasis of HCC was analyzed,and the phenomenon that TGF-?1promoted the invasion and metastasis of HCC by inducing the transformation of HSC to CAF in clinical specimens was verified.To establish an in vitro cell model simulating the microenvironment of hepatocellular carcinoma,human hepatoma cell lines BEL-7402,HepG2 and SMMC-7721,with different metastatic potential were co-cultured with hepatic stellate cell HSC,and TGF-?1 or TGF-?1 inhibitor was added to the culture medium to observe the morphological and cytoskeletal ultrastructural changes of HSCs treated with TGF-?1.The invasion and migration ability of hepatoma cells with different metastatic potential were observed.To further detect the expression of TGF-?1,CAF related marker molecules(?-SMA,FAP)in HSC treated with TGF-? 1,to study the phenomenon of TGF-?1 promoting the hepatocellular carcinoma metastasis and invasion by inducing the transformation of HSCs to CAFs.RESULTThe positive expression rates of TGF-?1,?-SMA and FAP in HCC tissues were higher than those in normal liver tissues,and the positive expressions of TGF-?1,?-SMA and FAP were related to the clinical metastasis of HCC.The results of Kaplan-Meier survival analysis showed that the survival time of patients with positive expression of TGF ? 1,?-SMA and FAP was decreased.After co-culture with hepatoma cells,LX2 cells changed from star-shaped or polygonal to spindle-shaped and flat-long fusiform,showing the morphological characteristics of myofibroblasts.after co-culture of LX2 cells with hepatoma cells,the expression of ?-SMA or FAP was significantly increased,TGF-?1 could significantly promote the expression of ?-SMA and FAP at gene and protein level,and TGF-?1 inhibitor could inhibit the above effects.After co-culture with LX2 cells,the migration and invasion ability of HCC cells increased significantly,and TGF-?1could significantly increase the migration and invasion ability of HCC cells,which could be abolished by the inhibitor of TGF-?1.After co-culture with LX2 cells,the expression of epithelial markers was significantly decreased,the expression of interstitial markers was significantly increased,and the expression of transcription factor Twist,which regulates the process of EMT,was significantly increased.The expression of tumor metastasis related molecules was significantly increased.After treatment with different concentrations of TGF-?1 and its inhibitors,TGF-?1 could inhibit the expression of epithelial markers,significantly promote the expression of interstitial markers and promote the expression of tumor metastasis related molecules.Inhibitors can cancel the effect of TGF-?1.CONCLUSION1.the experiment of human tumor tissue specimens confirmed that the expression of TGF-?1 and CAF markers ?-SMA and FAP in the stroma of HCC was increased,which was related to the clinical metastasis of HCC,and the survival time of patients with HCC with positive expression of TGF ? 1,?-SMA and FAP was shorter.2.Cell experiments in vitro confirmed that TGF-?1 could promote the transformation of hepatic stellate cells into cancer associated fibroblasts,and further promote the epithelial-mesenchymal transformation of hepatoma cells,thus promoting the metastasis and invasion of hepatoma cells.