TMPRSS4Induces Invasion By Facilitating EMT In Hepatocellular Carcinoma

Object:Postoperative recurrence,invasion and metastasis are related to the survival of patients of HCC. Some researches indicated that the epithelial mesenchymal transitions (EMT) is closely related to the metastasis of HCC without illustrating its mechanism. Our previous work showed that in different metastatic potential of hepatocellular carcinoma tissue, the expressions of TMPRSS4and EMT-related gene products such as E-cadherin, vimentin and fibronectin were different. In hepatocellular carcinoma tissue where the expressions of TMPRSS4were high, the expression of E-cadherin decreased and the expression of vimentin and fibronectin increased. It indicated that TMPRSS4may be related to the invasion and EMT in hepatocellular carcinoma. This study, on the foundation of our preliminary works, focused on the role of TMPRSS4in EMT of hepatocellular carcinoma and its mechanisms. We used the lentiviral to regulate the expression TMPRSS4in the BEL-7402cells, and to observe the influence of the overexpression of TMPRSS4on BEL-7402cells by detecting its invasion and metastasis and the expressions of EMT-related proteins. Then we could deduce the mechanism of TMPRSS4in EMT of hepatocellular carcinoma. And it would provide a new method for predicting and preventing the recurrence, invasion and metastasis of hepatocellular carcinoma.Methods:1. To construct the cell line: We constructed the lentiviral vector LV-TMPRSS4, and transferred it into the HCC cell line BEL-7402to establish over-expression of TMPRSS4in BEL-7402.2. Experimental groups:Three groups separately were normal BEL-7402control group (control), LV-GFP-empty vector control group (LV-GFP), TMPRSS4overexpression group (transfected LV-TMPRSS4vector, LV-TMPRSS4).3. DetectionWe observed the morphological changes of the BEL-7402hepatoma cells after they were transfected with lentiviral vector LV-TMPRSS4, using RT-PCR and Western Blot to detect TMPRSS4expression after transfection. Using cck-8, Transwell, Western Blot and other technologies, we explored the changes of proliferation, invasion potential, and expressions of relative EMT genetic products (eg. E-cadherin、Vimentin、Fibronectin) in BEL-7402hepatoma cells with TMPRSS4over-expression.Results:1. The expression of TMPRSS4enhanced after transfectionThe results of RT-PCR displayed that mRNA expressions of TMPRSS4in LV-TMPRSS4group increased after transfection. The results of Western Blot showed that, compared with the control group and LV-GFP group, the protein expression of TMPRSS4in LV-TMPRSS4group was significantly increased, while the control group and LV-GFP group had no significant difference in protein expression of TMPRSS4. The results indicated that cell line of BEL-7402had been transfected with the gene TMPRSS4successfully.2. The overexpression of TMPRSS4changed the morphology of BEL-7402After BEL-7402hepatoma cells transfected with LV-TMPRSS4, the morphological changes of cells were obvious from the original oval to spindle.3. Cell proliferation did not change in transfected cellsCCK-8results showed that, compared with the control group and LV-GFP group, the proliferation of LV-TMPRSS4group was not significant (P>0.05). It meant that the overexpression of TMPRSS4had no significant effect on on BEL-7402cells.4. The ability of cell migration after overexpressing of TMPRSS4in BEL-7402cells significantly enhanced Cell scratch assay in migration of three groups of cells, showed LV-TMPRSS4over-expression of cell migration was significantly strengthened, while there was no significant difference between the control group and the LV-GFP empty vector.5. The capacity of cell invasion enhanced in overexpression of TMPRSS4of BEL-7402Experiments of transwell displayed the number of cells passing through the Matrigel and reaching the back of Transwell membrane were44.0±2.6,which was more than the control group(15.7±2.1) and the LV-GFP group(16.7±2.1, P<0.05). It indicated that the invasion enhanced after transfection.6. The expression of EMT-related proteins after transfectionThe results of Western Blot showed that, compared with the control group and LV-GFP group, the expression of E-cadherin was downregulated, while the expression of Vimentin and Fibronectin were upregulated.Conclusion:1. The overexpression of TMPRSS4would change the morphology of BEL-7402cells into shuttle type, which makes it easier for the invasion and metastasis of tumor cells.2. The overexpression of TMPRSS4would significantly enhance the invasion and migration of BEL-7402cells, but had no effects on cell proliferation.3. The overexpression of TMPRSS4induced EMT (epithelial-mesenchymal transition) in BEL-7402cells. The expression of E-cadherin downregulated and the exopression of Vimentin and Fibronectin upregulated.4. TMPRSS4is closely related to the invasion and metastasis of HCC, and it will be a potential target for the treatment of liver cancer.