Mechanisms Of The Hepatoma Cell Differentiation Induced By Hepatocyte Nuclear Factor 4α

【Background and Objective】Hepatocellular carcinoma (HCC), as one of the most common malignant tumors worldwide, is high prevalent in our country. Most of the patients who develop this tumor have no symptoms and are failed to be diagnosed at an early phase. And, because of its high malignancy and invasivity, HCC is a major global health problem and it need to be treated with new potential therapies. Differentiation therapy, which opened a new era of treatments of cancer in the past two decades, is an approach to the treatment of malignancies so that the tumor cells can differentiate into mature cells and resume the process of maturation, leading to the suppression of the cell proliferation. Our previous studies have shown that hepatocyte nuclear factor 4α(HNF4α), a liver-enriched transcription factor, is expressed much higher in normal hepatocytes than in hepatoma cells. We used an adenovirus mediated gene delivery system to efficiently transfer HNF4αinto hepatoma cells, and the results demonstrated that the hepatoma cells with forced re-expression of HNF4αwere induced to differentiate into hepatocytes and result in up-regulation of a cluster of hepatocyte marker gene expression. Meanwhile, infection of hepatoma cells by HNF4αabolished their tumorigenesis in mice. Moreover, intratumoral injection of adenovirus carrying the HNF4αgene displayed significant antitumor effects on transplanted tumor models.To study the mechanisms of the hepatic differentiation effect by HNF4α, we used the cDNA microarray to detect differential gene expression profiles of Hep3B infected with AdHNF4αand AdGFP. In addition, the levels of mRNA expression and protein expression of a part of the listed genes in liver tumor cells and tissues were determined by real time RT-PCR, immunocytochemistry (ICC) and immunohistochemistry (IHC). There were 3 main parts in the present study, 1) differential gene expression profiles of Hep3B infected with AdHNF4αand AdGFP; 2) the reverse effects of HNF4αon epithelial to mesenchymal transition (EMT) in hepatoma cells; and 3) the inhibition of Wnt signaling pathway by HNF4αin hepatoma cells.【Methods】1. Differential gene expression profile of Hep3B infected with AdHNF4αand AdGFP Hepatoma cell lines Hep3B cells were seeded onto 35 mm culture dishes (5×10~5/dish) and infected with AdHNF4αand AdGFP (as a control) at MOI 100. The cells were collected 48 h and 72 h after infection. The total RNA was extracted and prepared for cDNA probes. Four cDNA microarrays (Affymetrix U133 plus 2.0 Array) were used to generate the differential gene expression profiles, and the image procession and data analysis were performed with Affymetrix GineChip Scanner 3000 and GeneChip Operating Software (GCOS). The expression of some listed genes was confirmed by real time RT-PCR, such as several EMT markers (TGFBR2, SMAD5, FSP1 and N-Cadherin) and a few Wnt pathway components (DKK4, FZD2, FRAT1 and AXIN1).2. Reverse effects on EMT in hepatoma cells by HNF4αReal time RT-PCR was carried out to detect the expression level of some EMT markers (TGFBR2, SMAD5, N-Cadherin, FSP1, E-Cadherin, Vimentin, Fibronectin 1, plakoglobin, ZO-1, TGFβ1 and SMAD2) in hepatoma cell lines Hep3B and HepG2. ICC was used to examine the location and expression of E-Cadherin, Vimentin and TGFβ1. Male nude mice were used for a subcutaneous tumor model. After intratumoral injection with AdCMV-HNF4αand AdCMV-GFP for 4 times, the nude mice were sacrificed and subcutaneous tumor tissues were obtained for the detection of the epithelial marker E-Cadherin and the mesenchymal marker Vimentin by IHC.3. Inhibition of Wnt signaling pathway in hepatoma cells by HNF4αSeveral Wnt signaling pathway components (DKK4, FZD2, FRAT1, AXIN1,β-catenin, GSK-3β, DSH) were quantified in mRNA level by real time RT-PCR. And the methods of ICC and IHC were used to determine the expression of DKK4 andβ-catenin in hepatoma cell lines (Hep3B and HepG2) and hepatoma tissues respectively.【Results】1. AdHNF4αinduces a cluster of the differential gene expression in Hep3B Differential gene expression profiles: 363 genes up-regulated and 435 ones down-regulated at 48h; and 1,058 genes up-regulated and 1,375 ones down-regulated at 72h in Hep3B cells after infection with AdHNF4α, respectively. The molecular functions of up-regulated genes in both time points are mainly related to protein binding, catalytic activity, transferase activity and transcription regulator activity, which are involved in the metabolism, regulation of biological process, regulation of cellular process, cell communication, development and so on. On the other hand, the down-regulated genes are related to the catalytic activity, enzyme regulation and oxidoreductase activity, which are participated in the biological processes such as development, localization and transport.Real time RT-PCR revealed tha the mRNA expression of some EMT markers (TGFBR2, SMAD5, FSP1 and N-Cadherin) and Wnt signaling pathway components (DKK4, FZD2, FRAT1 and AXIN1) was in accordance with that of cDNA arrays.2. HNF4αreverses the process of the EMT in hepatoma cells Differential expression of the selected genes related to EMT (TGFBR2, SMAD5, FSP1 and N-Cadherin) was confirmed by real time RT-PCR both in Hep3B and HepG2 after infection with AdHNF4αand AdGFP. Meanwhile, significant differential expression of some other EMT markers (E-Cadherin, Vimentin, Fibronectin 1, plakoglobin, ZO-1, TGFβ1 and SMAD2) was examined in Hep3B or HepG2, indicating the EMT process in hepatoma cells was reversed after AdHNF4αinfection. ICC results confirmed the up-regulation of E-Cadherin and the down-regulation of TGFβ1 both in Hep3B and HepG2 after infection with AdHNF4α, while Vimentin was down-regulated in Hep3B only. In addition, the up-regulation of E-Cadherin and the down-regulation of Vimentin were examined with IHC in the subcutaneous tumor tissues. All the results demonstrated the EMT process in hepatoma cells was reversed by HNF4α.3. HNF4αinhibits Wnt signaling pathway in hepatoma cellsThe gene expression of the major Wnt signaling pathway components deteced by real time RT-PCR was in accordance with that of cDNA arrays. ICC results showed that HNF4αcould induce DKK4 expression and reduceβ-catenin expression in hepatoma cell lines Hep3B and HepG2. Moreover, the localization ofβ-catenin was altered from the nuclei and cytoplasm to the cytoplasmic membrane significantly in Hep3B. Finally, with the method IHC, we revealed that DKK4 was up-regulated whileβ-catenin was down-regulated in the subcutaneous tumor tissues after injection of AdHNF4αas well. The present study indicates HNF4αinhibits Wnt signaling pathway.【Conclusion】1. HNF4αinduces the differentiation of hepatoma cells into hepatocytes and a cluster of differentially expressed genes is involved in the metabolism, development and cellular signaling transduction, etc.2. HNF4αreverses the EMT process in hepatoma cells.3. HNF4αinhibits the Wnt signaling pathway, which presents as the major mechanism of the hepatic differentiation effects of HNF4α.