Prevention Of Hepatocellular Carcinoma Development In Rats By Hepatocyte Nuclear Factors 4α And Its Molecular Mechanism

【Background and Objective】Primary liver cancer is the fifth most common cancer in the world and the third most common cause of cancer mortality.Hepatocellular carcinoma(HCC) accounts for between 85%and 90%of primary liver cancers.The diagnosis and treatment of liver cancer have witnessed major changes and the outcome for patients has significantly improved over the past decades.However,HCC remains a big burden as there are still numerous patients with chronic hepatitis and cirrhosis who are at the high fish for HCC.Therefore,the prevention of HCC is still on the hotspot worldwide.Hepatocyte nuclear factor 4(HNF4) is a member of the nuclear hormone receptor family of transcription factors.HNF4αis a main phenotype of HNF4 which plays an important role not only in regulating hepatocyte differentiation and maintaining liver-specific functions including energy metabolism,xenobiotic detoxification,bile acid synthesis,serum protein production,but also in controlling epithelial phenotype of hepatocyte.Importantly,HNF4αregulates the expression of genes encoding proteins involved in all major types of cell junctions including tight junctions,adhesion junctions, gap junctions,desmosomes,as well as epithelial polarization and cytoskeletal organization.Epithelial-to-mesenchymal transition(EMT) is the process that epithelial cells gradually lose their epithelial signatures while acquiring the characteristics of mesenchymal cells in cell morphology,structure,biological function,adhesion and migration ability.Recent studies revealed that EMT is not only involved in invasion and metastasis of cancer,but also implicated in the generation of cancer stem cells(CSCs) and tumorigenesis.It has been well accepted that Wnt/β-catenin signaling cascade is responsible for the proliferation,differentiation and migration of cells including hepatocyte.β-catenin is not only an important component in cell-cell adhesion but also a key molecular in Wnt signaling pathway.Along with the EMT,aberrant activation of Wnt/β-catenin(i.e.the accumulation ofβ-catenin in cytosol or the nuclear translocation) is usually observed in liver fibrosis and hepatocarcinogenesis.In the present study,we will investigate the role of HNF4αin the prevention of chemically induced HCC and further clarify the underlying molecular mechanism,which will lay the foundation for the clinical study of HNF4α.【Methods】1.Effect of HNF4αon experimental hepatocarcinogenesisThe replication-deficient recombinant adenoviruses carring HNF4αgene(AdHNF4α) and control adenovirus-AdGFP were packaged in 293 cells respectively.Then adenoviruses were stored after purification by cesium chloride gradient centrifugation and determination of the viral titers.HCC models induced by diethylinitrosamine(DEN):Fifty male Wistar rats were divided into 4 groups randomly.Group 1(n=12) was served as a normal control,and the rats in next three groups(n=50) were hepatic carcinogenesis models induced by DEN(70 mg/kg) once per week for 10 weeks intraperitoneally.The second group was served as model group and the third group was served as AdGFP control group.The last group was served as HNF4αtreatment group.Rats were infused with same amount of saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein,respectively at the time point of 10 w,12 w,14 w,16 w,18 w and 20 w.The animals were sacrificed and subjected to morphological and histological study at the end of 22 w.Immunohistochemical staining was used to detectα-SMA,AFP and PCNA.For the semiquantitative analysis,stained sections were measured by an image analyzer.Masson's trichrome and Sirius Red staining was utilized to detect the expression of collagens.For the semiquantitative analysis,the red-stained areas in the Sirius Red stained sections and blue-stained connective tissues in Masson's trichrome stained sections were measured on an image analyzer by a technician blinded to the samples.2.Inhibitory effect of HNF4αon hepatocyte EMT and CSC-like cells generation.Real-time RT-PCR and Imunohistochemistry staining assay were performed to detect HNF4αand EMT associated genes(E-cadherin,vimentin,snail,TGF-β1 ) in the rat livers of different group and tissue sample from HCC patients.Primary hepatocytes derived from male Wistar rats were cultured for 2 days after isolation,then the culture medium was replaced by serum-free medium containing 2 ng/ml TGFβ1.AdGFP or AdHNF4αat multiplicity of infection(MOI) 10 was added simultaneously with addition of TGFβ1.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,albumin(ALB),E-cadherin,Vimentin,Snail and collagenⅠmRNA in the cells was detected by real-time PCR respectively after adenovirus infection for 48 and 72 h. Indirect cytoimunochemistry assay was utilized to detect the expression of distribution of E-cadherin,vimentin and snail.Western blot assay was use to determine the protein level.Real time-PCR and immunohistochemistry were carried out to determine the transcript of liver progenitor cell/cancer stem cell associated genes including OV-6,EpCAM,Thy-1,CD133,c-kit.3.Regulation of Wnt/β-catenin signaling pathway by HNF4αLuciferase assay was used to analyze the inhibitory effect of HNF4αonβ-catenin transcription activity.Nuclear protein extraction followed by Western blot was utilized to detect the influence of HNF4αtranslocation ofβ-catenin triggered by TGF-β1. Immunohistochemical staining was performed to detect the expression and distribution ofβ-catenin in rat liver.【Results】1.HNF4αprevents the development of hepatocarcinogenesis in ratsLiver fibrosis,cirrhosis and HCC were observed in the process of DEN- induced rat hepatocarcinogenesis.In the 22-week,notable liver cirrohsis was found in the rats of model group and GFP control group,but in HNF4αgroup not.Moreover,all livers of model group and GFP control group exhibite neoplastic node,while there is no tumor node can be deteced in HNF4αgroup.Semi-quantitive analysis based on Masson's trichrome and Sirius Red staining showed the significant decrease of collagen in liver of HNF4αgroup copared with GFP group(49%).Collagen andα-SMA were significantly reduced in rat livers of AdHNF4αtreatment group compared with those of AdGFP treatment group. We also observed the increased expression of AFP and PCNA in livers of AdGFP infected rat,whereas AFP and PCNA expression in rats of AdHNF4αtreatment group were significantly decreased.2.HNF4αinhibits the EMT of hepatocyte and the generation of CSC-like cells Both real-time RT-PCR and immunohistochemistry showed the expression of HNF4αand epithelia cell marker E-cadherin gradually decreased during the development of hepatic fibrosis and hepatocarcinogenesis induced by DEN,whereas the mesenchymal cell marker Vimentin was notably increased(P＜0.05).More interestingly,compared with control groups AdGFP and saline;AdHNF4αinjection remarkly interrupted the up-regulation of E-cadherin and the down-regulation of Snail or Vimentin.Accompanied with the morphological change,decreased expression of both HNF4αand E-cadherin and increased expression of both vimentin and snail,which are the characteristic signatures of EMT,were observed in TGF-β1 treated primary liver cells. Compared with AdGFP infected cells,HNF4αintroduction recovered the morphology and the expression of E-cadherin,vimentin and snail of the primary liver cells,which strongly indicates the inhibitory effect of HNF4αon EMT of liver cells.Moreover,result from immunohistochemical assay showed that number of OV-6 and EpCAM positive cells, which are predicted as liver CSC,markedly enhanced during the process of DEN-induced experimental liver cancer.However,these CSC-like cells reduced with the treatment of Ad HNF4α.3.HNF4αsuppresses the signaling pathway of Wnt/β-cateninLuciferase assay demonstrated the inhibitory effect of AdHNF4αon theβ-catenin transcription activity in 293T,HepG2 and BRL cells on a dose-dependent manner. Western blot assay demonstrated that HNF4αwas able to repress TGF-β1-triggeredβ-catenin translocation,thus suppress the activation of Wnt/β-catenin signaling. Moreover,β-catenin was released from the membrane and accumulated in cycosol and nucleus in the process of DEN-induced hepatocarcinogenesis.Nevertheless,β-catenin translocation was significantly decreased in the liver of AdHNF4αtreated group compared with that from AdGFP control group(P＜0.05).【Conclusion】1.HNF4αexpression is reduced accompanied with EMT process during the development of hepatic fibrosis and hepatocarcinogenesis.2.HNF4αinjection remarkly inhibits the hepatic fibrosis and cirrhosis and prevents the generation of hepatocarcinogenesis in rats.3.Repression of the Wnt/β-catenin pathway activation is responsible for the inhibitory effect of HNF4αon EMT,CSC-like cells generation and hepatocarcinogenesis.