Study On The Inhibitory Effects Of Tfpi-2on Human Hepatocarcinoma Cells In Vitro And Vivo

Objective:To investigate the effects of tissue factor pathway inhibitor-2(TFPI-2) gene on the proliferation, apoptosis and expression of alpha fetoprotein of hepatocarcinoma cell line Hep3B. Furthermore, to investigate the inhibitory effects of TFPI-2on the growth and angiogenesis of human hepatocarcinoma cell Hep3B subcutaneous xenograft tumors in nude mice.Methods:The recombinant vector of pcDNA3.1-TFPI-2was transfected into Hep3B cells with liposome, and the cells were selected by G418. The expressions of TFPI-2mRNA and protein were detected by RT-PCR and Western blot respectively. The influence of TFPI-2on the proliferation of Hep3B cells via growth curve was assayed by CCK-8method. The colony-forming unit assay was used to measure single cell self-replication ability. The expression of AFP mRNA was detected by RT-PCR and the AFP secretion of Hep3B cell was measured by electro-chemiluminescence (ECL) immunoassay. Furthermore, the cell apoptosis was tested by flow cytometry. Then, the selected monoclonal Hep3B cells and untransfected Hep3B cells were transplanted into the subcutaneous tissue of nude mice respectively, and xenograft tumor volumes were measured every three days and the growth curves of tumors were drawn. The mice were killed after three weeks. The expressions of TFPI-2and VEGF mRNA and protein in tumor tissue were analyzed by qRT-PCR and Western blot respectively, the expression of TFPI-2protein and microvessel density (MVD) in xenograft tumors were observed by immunohistochemistrv.Results:In vitro, the expressions of TFPI-2mRNA and protein were identified in cells transfected by pcDNA3.1-TFPI-2. The growth rate and self-replication ability of Hep3B cells were significantly lower than those of the two control groups. The inhibition ratio of AFP mRNA expression was16.51%, and the AFP secretion of Hep3B cells transfected with pcDNA3.1-TFPI-2was significantly lower than the control groups (P<0.01). The rate of early apoptosis was significantly increased (24.03%±7.2%vs8.77%±3.66%). In vivo, the ultimate tumor volume of Hep3B-TFPI-2group was apparently smaller than those of2control groups (p<0.05). The relative expressions of TFPI-2mRNA and protein of Hep3B-TFPI-2group were significantly higher than those of the other groups (p<0.05); while, the relative expressions of VEGF mRNA and protein were apparently lower. Compared to2control groups, the inhibitory rate of VEGF protein expression were 19.8%and23.5%, respectively (p<0.05). The MVD in Hep3B-TFPI-2group was apparently lower than the control groups (p<0.05).Conclusions:The expression of TFPI-2can inhibit the growth and expression of AFP of hepatocarcinoma cells as well as induce early apoptosis. Futhermore, it can significantly inhibit the growth and angiogenesis of hepatocarcinoma cell Hep3B subcutaneous xenografts in nude mice.