Experimental Study On The Induction Of Transmembrane Protease Serine 4 In The Angiogenesis Of Hepatocellular Carcinoma

Objective:Hepatocellular Carcinoma(HCC)is one of the most malignant tumor in the world,as well as its incidence and mortality rank in the forefront of cancer.Exploring the mechanism involved in invasiveness and metastatic ability of HCC can be of great significance to early diagnosis and treatment,prolong the survival and improve the prognosis of HCC patients.Studies indicated that the staging of tumor is associated closely with angiogenesis,and anti-angiogenesis can be considered as an effective target for controlling tumors.Transmembrane serine protease 4(TMPRSS4)belongs to type ? transmembrane serine protease(TTSPs),with crystal structure of typical serine protease,promoting recurrence and metastasis of hepatocellular carcinoma.In the previous study,TMPRSS4 has been verificated that its ability of enhancing invasiveness and metastasis through inducing epithelial mesenchymal transitions(EMT)of hepatocellular carcinoma.However,both the effect of and its regulatory mechanism on angiogenesis remain unknown.Based on the above researches,we will further explore the function of TMPRSS4 on angiogenesis in hepatocullar carcinoma.The results of this study will provide a new therapeutic target for hepatocullar carcinoma.Methods:1.The cell line and experimental groups:Firstly,we constructed the lentiviral vector Lv-TMPRSS4,and transferred it into the HCC cell line,including MHCC97H and HuH-7 cell lines,to establish over-expression of TMPRSS4.There groups eparately were normal control group(control),Lv-GFP-empty vector control group(Lv-GFP),TMPRSS4 overexpression group(transfected Lv-TMPRSS4 vector,Lv-TMPRSS4).Then the nude mice were inoculated subcutaneously with Lv-GFP cells or Lv-TMPRSS4 cells.2.Detection:Matrigel-based tubular formation assay was used to detect the effect of TMPRSS4 on HCC angiogenesis,by the cell lines EA.hy926.CD34 was detected by immunohistochemistry reflecting the effect of TMPRSS4 on angiogenesis in the nude body.Then the RT-Realtime PCR was used to measure the expression of VEGF in hepatocellular cell lines with different TMPRSS4 level.The expressions of p-mTOR and mTOR were detected by western blot.Results:1.Construction of over-expression cell line:After lentiviral vector Lv-GFP and Lv-TMPRSS4 transfected in HCC cell lines including MHCC97H and HuH-7 for 48h,green fluorescence was observed in Lv-GFP group cells and Lv-TMPRSS4 group cells.The results of qRT-PCR?Western Blot showed that mRNA and protein expression of TMPRSS4 in Lv-TMPRSS4 group cells increased after transfection,which indicated that cell line MHCC97H and HuH-7 had been transfected with the gene TMPRSS4.2.The overexpression of TMPRSS4 enhanced vasculogenic in the Matrigel:The tube formation assay showed that vasculogenic mimicry formed by EA.hy926 increased with Lv-TMPRSS4 group cells supernatant compared control group cell and Lv-GFP group cells.The results indicated that some cytokine may be secreted by Lv-TMPRSS4 group cells which leaded to increased angiogenesis ability.3.The overexpression of TMPRSS4 enhanced angiogenesis in vivo:Immunohistochemistry result of CD34 indicated that microvesseldenisity(MVD)was higher in Lv-TMPRSS4 group.As a consequence In nude hepatocellular carcinoma model,the overexpression of TMPRSS4 enhanced angiogenesis.4.The expression of VEGF was upregulated in over-expression of TMPRSS4:Compared with the control group and Lv-GFP group,the expression of VEGF detected by qRT-PCR was upregulated.5.The expression of p-mTOR after transection:The results of Western Blot showed that,compared with the control group and Lv-GFP group,the expression of p-mTOR was upregulated,without significient change in total-mTOR.Conclusions:1.TMPRSS4 promotes angiogenesis in hepatocullar carcinoma in vitro and in vivo.2.TMPRSS4 promotes angiogenesis by up-regulating the expression of VEGF.3.TMPRSS4 enhances p-mTOR expression via activating mTOR signal pathway.