The Reasearch Of Effect And Mechanism Of PTEN On Human Hepatoc-arcinoma Cell In Vitro

Part1: Gene Therapy Targeting for Hepatocellular Carcinoma: Selective and Enhance the tumor Suppressor PTEN Gene Expression Regulated by a Human a-Fetoprotein Enhancer Linked to its PromoterSection A: Construct of a targeting gene therapy vector carrying AFP enhancer and promoter for expressing PTEN gene in AFP-producing hepatocarcinoma cellObjective: To construct a recombinant vector carrying abbreviated cis-acting element of AFP for specific expression of wild type PTEN gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells.Methods: Primers containing specific enzyme cutting sites were designed to amplify the DNA segments of minimum essential AFP enhancer and promoter and synthesized by polymerase chain reaction (PCR) from Genome DNA of HepG2 cells. The DNA fragments were inserted into the multiclone site (MCS) of promoterless PTEN vector, and then the recombinant plasmid named pAEF— AFP-PTEN carrying the 0.7 kb cis-acting element of AFP and wild-type PTEN gene was constructed.Results: The AFP enhancer/promoter was synthesized through PCR from the human genome. The cis-acting element of AFP were directly inserted into the promoterless plasmid carrying PTEN gene as confirmed by restriction digestion and DNA sequencing.Conclusion: The recombinant expression vector that carrying wt-PTEN gene guided by AFP enhancer and promoter was successfully constructed and it could be used as a gene therapy vector for HCC in vitro.Section B: The Targeted Expression of Recombinant Vector pAEF-AFP-PTEN in Hepatoma CellsObjective: To investigate the specific expression of the recombinant vector pAEF—AFP—PTEN in an AFP-expressioning hepatocellular carcinoma cell(HepG2).Methods: The recombinant plasmid was transfected into AFP positive or negative cell lines including two hepatocarcinoma cell line (HepG2,), a normal liver cell line (LO2), and a cervical cancer cell line (HeLa) by the means of lipofectamine. Immunofluorescence cytochemistry, reversely-transcripted PCR (RT-PCR), immunohistochemistry and Western blotting were used to detect the expression of the PTEN gene at mRNA or protein level in four different cell lines.Results: RT-PCR, immunohistochemistry and Western blot showed that PTEN gene was markedly transcribed in the AFP-expressioning hepatocarcinoma cell line HepG2, but only slightly in non-AFP-expressing cell lines, LO2 and HeLa cells. In addition, strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy, but fluorescence was very weak in LO2 and HeLa cells.Conclusion: Under control of the cis-acting element of human AFP, the recombinant vector carrying PTEN gene can be specially expressed in AFP-producing HepG2 cells. Therefore, this plasmid system can be used as a novel, potent and specific tool for gene-targeting therapy for the AFP positive primary hepatocellular carcinoma. Part2: Effects of the Tumor Suppressor Gene PTEN on Biological Behaviour in Human Hepatocar cinomaCell Line HepG2Objective: To evaluate the effects of exogenous plasmid-mediated gene transfer of human tumor suppressor gene PTEN on biological behaviour of prolifiration, adhesion, migration and invasion ablility in human hepatocarc-inoma cell line HepG2 in vitro.Methods: The recombinant expression vector carrying wild-type PTEN gene constructed by ourself was transducted with lipofectamine into the HepG2 cells. The change of cell apoptosis was measured by flow cytometry and electronic microscope. MTT assay was used to assess the effect of PTEN on proliferation. The in vitro migration of HepG2 cells transfected by the recombinant plasmid on extracellular matrix matrigel was assayed with cellular migration test and the invasive abilities in vitro detected by Transewll chamber were also examined; further, the effect of adriamycin on the transfected cells were detected by MTT. In all these assays, pBp was used as control.Results: The growth of HepG2 cells treated with PTEN was significantly inhibited. Some classical morphological changes of apoptosis appeared under electronic microscope. After PTEN infected, HepG2 cells showed a significantly increase in rates of apoptosis in comparison with controls(P<0.05=. Meanwhile, the in vitro cell adhesion experiment showed that the PTEN-HepG2 cells decreased cell adhesion to matrigel and fibnectin extracellular matrix. The invasive ability of liver cancer cell line was inhibited by expression of PTEN effectively. PTEN can also increase chemosensitivity of human hepatoma.and the 50% inhibitory concentration(IC50) of ADM was reduced in transfection cells (P<0.01)Conclusion: Plasmid mediated expression of human tumor suppressor gene PTEN has an inhibitory effect on proliferation and invasive ability of human liver cancer cell lines HepG2. PTEN can reverse the ADM resistance of HCC cells in some degree.It may become a new gene therapeutic agent for HCC. Part3: The preliminary research of inhibitory mechanism of PTEN in hepatocarcinoma cellsObjective: To investigate the preliminary research of inhibitiory mechanism of PTEN in hepatocarcinoma cells.Methods: The expression of PTEN, pAkt, NF-κB and their mRNA were detected by Immunohistochemical staining and RT-PCR in HCC and their pericarcinomatous tissues respectively, furthermore the combination of their expressions with the tumor's clinicopathological characteristic and invasion potential were studied. HepG2 cells transfected or not was exposured to PI3K specific inhibitor LY294002 with or without followed NF- κB specific inhibitor Pyrrolidine dithiocarbamate (PDTC) in vitro. RT-PCR and Western blot were used to determin any changes of mRNA and protein of PTEN, pAkt, NF-κB in different conditions. EMSA (Electrophoretic Mobility Shift Assay) supershift assays and LCM (Laser-Scaning Confocal Microscope) were also employed to assess the binding of NF-kB activity and protein level in nucleus.Results: The positive mRNA and protein expression of PTEN in HCC was remarkably lower than that in pericarcinomatous liver tissues, while the positive expression of pAkt, NF- κB was both higher in pericarcinomatous liver tissues (P<0.05). Moreover, there was negatively relationship between the expression of PTEN and that of, whereas that of between pAkt and NF- κB was entirely contrary. we found that LY294002 significantly inhibited both pAkt and NF-κB activity, while the specific NF-κB inhibitors (PDTC) had no effect on pAkt following LY294002 exposure. However, HepG2 transfection with PTEN (wild-PTEN) gene failed to have any changes of pAkt and NF-κB stimulated by LY294002 and PDTC.Conclusion: The tumor suppressor PTEN may function in growth inhibiting process of the hepatoma carcinoma cell through antagonize the pAkt-NF-κB signaling cascade. This study provides an rationale for the further development of PTEN as a potential therapeutic tool in the gene therapy of liver cancer.