The Role Of Tumor Suppressor Gene Tg737in The Pathogenesis Of Hepatocellular Carcinoma And The Underlying Molecular Mechanisms

[Background]Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, isamong the most common malignancies, with an increasing incidence in China. Itsmortality rate ranks the third place after stomach cancer and esophagus cancer. Despitethere are some improvements in diagnosis and therapy of HCC, it still remains bigproblem because the clarification of molecular mechanisms responsible for HCCpathogenesis is unclear. It is of paramount importance to elucidate the the occurrence andprogression of HCC, not only for an understanding of tumor biology but also to permit the development of specific therapies that effectively target HCC.Tg737, a new kind of tumor suppressor gene in HCC, was originally identified byinsertional mutagenesis and positional cloning as the gene which is altered in autosomalrecessive polycystic kidney disease in the TgN737Rpw transgenic mouse line. The Tg737gene encodes an824amino acid protein which includes10copies of a34-amino acidtetratrico-peptide repeat motif. Some previous studies provided evidence that Tg737mayplay an important role in carcinogenesis of kidney and pancreas. However, the role oftumor suppressor gene Tg737in the pathogenesis of HCC and the underlying molecularmechanisms remain poorly understood.[Aims]In order to effectively investigate whether Tg737may function in the malignanttransformation of fetal liver stem/progenitor cells (FLSPCs) and underlying molecularmechanisms, we first improved the differention method. To induce hepatic differentiation,bone mesenchymal stem cells (BMSCs) and FLSPCs were cultured using dynamiccultured scaffold (DCS), furthermore, this improved differention method was used insubsequent related experiments. Then, to investigate the role of Tg737signaling pathwayin in the pathogenesis of HCC and the underlying cellular and molecular mechanisms. Toexplore new melecular mechanisms regulating HCC pathogenesis and provide newtheoretical basis for HCC prevention and therapy.[Methods]1. A modified protocol previously reported was used to obtaine the decellularized liver.In this study, the identified murine BMSCs and FLSPCs were randomized into2groupsrespecticely. We compared the hepatic differentiation of BMSCs and FLSPCs in abiomatrix scaffold from rat liver (dynamic cultured scaffold (DCS) and in the presence ofhepatocyte growth factor (HGF)) with a two-dimensional substrate(adherent culture and inthe presence of HGF). At the indictaed time of cell growth, cells were harvested by trypsindigestion and then the expression of hepatocyte-specific genes alpha fetoprotein (AFP),albumin (ALB), forkhead box protein A1(FOXA1), hepatic nuclear factor4alpha (HNF4α)and cytochrome P4501A2(CYP1A2) were measured by western blot.2. Tg737inhibition was achieved by short hairpin RNA (shRNA). StableshRNA-expressing clones (Tg737-silent FLSPCs, sFLSPCs) were selected by puromycindihydrochloride. To select cells expressing high levels of stem cell marker CD133for usein the next study, first step in this part was purification of Tg737normal FLSPCs (nFLSPCs) and sFLSPCs by PCGC. Then, CD133were analyzed by flow cytometry at1,7and14days during the culture period. Following RNAi of Tg737, the mRNA andprotein levels of sFLSPCs were measured by PCR, western blot andimmunocytochemistry analyses. The celluar growth curve was determined using cellcounting and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The proliferative capacity studies were determined by flow cytometry, including thecellular proliferation marker Ki-67expression analysis, cell cycle and apoptosis. Atindicated time of cell growth in the presence of HGF, enzyme linked immunosorbent assay(ELISA) was used for quantifying the ALB and AFP secreted by nFLSPCs and sFLSPCs.After incubation of nFLSPCs and sFLSPCs in complete medium supplemented with HGFfor7days, cells were harvested for fine structure analysis. nFLSPCs and sFLSPCs werealso seeded into scaffold for DCS, after7days in culture with complete mediumsupplemented with HGF, western blot was performed to measure AFP, ALB, FOXA1,HNF4α and CYP1A2expression. The invasion ability was evaluated by transwell assay.The in vivo tumorigenesis was detected by injection nFLSPCs and sFLSPCs intosubcutaneous of athymic mice. In order to investigate whether whether cyclin D1andcyclin B were involved in the effects of Tg737on cell proliferation and cell cycleprogression, western blot analysis was performed. Using western blot andimmunocytochemistry, we also analyzed the synergy of Tg737and HNF4α in thedifferentiation of FLSPCs.3. HepG2and MHCC97-H cells were routinely cultured in Dulbecco’s Modified EagleMedium (DMEM) supplemented with10%fetal bovine serum (FBS). In all subsequentrelated experiments, the cells were treated with medium supplemented with1%FBS,unless otherwise noted. For the incubation of cells under hypoxic conditions, the cellswere exposed to1%O2with5%CO2at37°C for the indicated times. To exclude thepossibility of apoptosis-related effects caused by low-serum medium under normoxic orhypoxic conditions in subsequent experiments, Annexin V/propidium iodide (PI) assayswere performed. HepG2and MHCC97-H cells were subjected to normoxic or hypoxicconditions, and the cell adhesion, invasion and migration capabilities were evaluated. Theexpression of Tg737under normoxia or hypoxia was detected using western blot.Furthermore, we created HepG2and MHCC97-H cells that overexpressed Tg737prior toincubation under hypoxia and investigated their metastatic characteristics. Finally, weanalyzed the involvement of critical molecular events (polycystin-1, interleukin-8(IL-8) and transforming growth factor β1(TGF-β1)) known to regulate invasion and migration.[Results]1. A systematic method for BMSCs enrichment used in this study can effectively enrichand purify BMSCs. The harvested BMSCs are in a good state, highly express stem cellsmarkers CD44, low expression of hematopoietic stem cell markers CD45. The enrichedFLSPCs were with small size, larger nuclear/plasma, highly express stem cells markersCD49f and CD133, low expression of mature liver markers. The isolated FLSPCs couldgenerate mature ALB expressing hepatocytes. In the presence of HGF, DCS could betterstimulate the BMSCs to express endodermal and hepatocyte-specific genes and proteinsassociated with improved functions, and the cells exhibited the ultrastructuralcharacteristics of mature hepatocytes. When transplanted into acute liver injured mice,DCS plus GF cultured cells exhibited increased liver function, survival, engraftment intothe host liver and further hepatic differentiation. Compared to the adherent with HGFcultured FLSPCs, the combination of DCS and HGF could better better induce FLSPCs todifferentiation into hepatocyte-like cells. This improved differentiation method can beused in subsequent related experiments.2. The microscopic observations of freshly purified nFLSPCs and sFLSPCs revealed nosignificant morphological changes in sFLSPCs. Over the course of cell culture, on day1nFLSPCs and sFLSPCs showed high expression levels of CD133and there was nosignificant difference between the two groups; with time, the expression of CD133gradually decreased in both groups. CD133expression was significantly higher insFLSPCs than in nFLSPCs at all time points sampled except day1. Following Tg737inhibition, the mRNA and protein levels of sFLSPCs decreased as shown by PCR, westernblot and immunocytochemistry analyses. Excluding apoptosis-related effects, we foundthat Tg737inhibition resulted in enhanced cell proliferation through promoting cell-cycleprogression. Tg737inhibition also resulted in significant arrest of cell differentiation,stable expression of both ALB and AFP and quiescent ultrastructure. The improveddifferentiation method DCS plus HGF also confirmed differentiation arrest. Furthermore,the FLSPCs with Tg737inhibition got strong invasion and hepatocarcinogenesis ability.Cyclin D1and cyclin B are important for cell growth and are key signaling proteins in cellcycle progression. Silencing of Tg737induced increases in cyclin D1and cyclin B proteinlevels. Following downregulation of Tg737, the level of HNF4α decreased; these twoproteins co-localized in FLSPCs. 3. The treatment of HepG2and MHCC97-H cells with low-serum medium undernormoxic or hypoxic conditions did not significantly affect cell viability in vitro. Exposureof these two HCC cell lines to hypoxic conditions reduced HCC cell adhesion andfacilitated invasion and migration. In this study, Tg737expression was significantlyinhibited in HepG2and MHCC97-H cells following exposure to hypoxia. Thedownregulation of Tg737expression corresponded to significantly decreased adhesion andincreased invasion and migration. The treatment of cells with low-serum medium undernormoxia did not significantly affect Tg737expression. Hypoxia also decreased theexpression/secretion of polycystin-1, increased the IL-8, and increased the levels of activeand total TGF-β1. Moreover, the decrease in adhesiveness and the increase in the invasiveand migratory capacities of hypoxia-treated hepatoma cells were attenuated bypcDNA3.1-Tg737transfection prior to hypoxia. Finally, following the upregulation ofTg737, the expression/secretion of polycystin-1increased, and the secretion of IL-8andthe levels of active and total TGF-β1decreased correspondingly. Cell viability,liposome/pcDNA3.1(-) had no effects in our study.[Conclusions]1. An effective method for hepatic differentiation of BMSCs and FLSPCs issuccessfully constructed. The combination of DCS and HGF could better promote theBMSCs and FLSPCs to mature into functional HNCs. This improved differentiationmethod can be used in related experiments of FLSPCs.2. Tg737inhibition by shRNA in FLSPCs leads to malignant transformation throughincreasing proliferation by accelerating cell-cycle progression and concomitantdifferentiation arrest, furthermore, cyclin D1, cyclin B and HNF4α signaling pathway maybe an important intermediary in this process.3. Tg737contributes to hypoxia-induced invasion and migration, partially through thepolycystin-1, IL-8, and TGF-β1pathway.