| ?Background and Objective?Up to now, hepatocellular carcinoma(HCC) is still one of the most common malignant tumors globally. And it is also the most common causes of death among all kinds of the malignant tumors. The southeast of Asia, including China, is the most heavy striken area of HCC. Despite the clinical implementation of numerous therapeutic strategies, the 5-year survival of HCC still remains low. Metastasis is the main risk for the long-term survival for HCC patients after surgical treatment and is considered to be responsible for the high HCC mortality rate. HCC metastasis is a complex cascade of events that include multiple genetic alterations. In the last few years, epithelial–mesenchymal transition(EMT) has been thought to play a critical role in HCC metastasis. There are several pathways that have emerged as important regulatory signals for EMT, including the PI3-K/Akt-, Wnt-, Notch-, Hedgehogand nuclear factor-kappaB-dependent pathways. However, the mechanisms underlying the metastasis of HCC have not been thoroughly understood.The hepatocyte nuclear factor(HNF) family, which consists of HNF1, HNF3, HNF4, HNF6 and CCAAT/enhancer binding proteins, form a systemic transcriptional network critical for the development and functional maintenance of hepatocytes in the liver. More and more evidence has suggested that the suppression and genetic alterations of HNFs are involved in liver tumorigenesis. Our previous studies demonstrated that the expression of both HNF4? and HNF1? were markedly reduced in most of the human HCC tissues. Upregulation of either HNF4? or HNF1? induced the differentiation of hepatoma cells into more mature phenotypes, abolished their tumorigenesis and inhibited the growth of HCC xenograft in mice. Moreover, we also showed that overexpression of HNF4? could ameliorate hepatic fibrosis and liver function with the inhibition of EMT in both hepatocytes and activated hepatic stellate cells in rats. Interestingly, a study by Hatziapostolou et al. reveals that even transient inhibition of HNF4? in hepatocytes will initiate hepatocellular transformation in vitro and in vivo through a microRNA inflmmatory feedback circuit. More recently, Walesky et al. demonstrated that HNF4? deletion in adult hepatocytes leads to increased progression of HCC in mice by using a tamoxifen-inducible Cre recombinase. All these data suggest that HNFs, the differentiation-determining transcription factors for hepatocytes, are central regulators of hepatocarcinogenesis and highlight HNFs as potential therapeutic targets for HCC.The forkhead box transcription factor A(FOXA, also known as HNF3) consists of FOXA1(HNF3?), FOXA2(HNF3?) and FOXA3(HNF3?). Experiments examining embryos defiient for both FOXA1 and FOXA2 genes indicated that both genes are required for hepatic specifiation. It has also been proven that FOXA2 is essential for glucose and lipid homeostasis in the liver. Early studies showed the expression of FOXA2 is unchanged in mouse liver tumor and even increased in human HCC. However, the expression of FOXA2 was suppressed in several other kinds of tumors, including lung, breast and thyroid cancers. More recently, the role of FOXA2 in tumor metastasis has also been described. Loss of FOXA2 expression promoted EMT through regulation of E-cadherin and promoted metastasis of pancreatic cancer. More interestingly, the inactivation of FOXA2 through IKK?-mediated phosphorylation contributed to inflmmation-mediated liver tumorigenesis. FOXA1 and FOXA2 were essential for sexual dimorphic HCC in mice. These data indicate that FOXA2 functions as a suppressor in HCC. Nevertheless, the expression levels of FOXA2 in human HCC have not been evaluated and the role of FOXA2 in HCC metastasis remains obscure.In this study, we explored the expression of FOXA2 in human HCCs and its clinical signifiance. The role of FOXA2 in HCC metastasis and the potential mechanism were also addressed.?Methods?1. The expression of FOXA2 in human HCC tissues, adjacent normal tissues and portal vein tumor thrombus(PVTT) tissuesThe expressions of FOXA2 in 72 human HCC tissues, adjacent normal tissues and portal vein tumor thrombus(PVTT) tissues were examined by Real-time PCR and Western blot. We also compared the FOXA2 expression in human HCC tissues with venous invasion(HCC-VI) and without venous invasion(HCC). Then, the relationship between FOXA2 expression and the clinicopathologic characteristics of HCC patients was analyzed.2. Construction of lentivirus that overexpression of FOXA2 and downexpression of FOXA2The backbone plasmid pSin-EF1?-FOXA2 for producing FOXA2-overexpressi ng-lentivirus lenti-FOXA2 has been successfully constructed by our lab member Dr. Chang-Peng Zhu previously. And the effect of pSin-EF1?-FOXA2 in HCC ce lls has been tested. The FOXA2 knockdown lentivirus system consists of three p lasmids, including pLKO.1, pMD2.G and psPAX2, which were purchased from A ddgene. The single-chain DNA oligos containing the FOXA2 shRNA sequence fl anked by sequences that are compatible with the sticky ends of EcoRI and Age I were synthecized. Forward and reverse oligos are annealed and ligated into the pLKO.1 vector, producing the final plasmid pLKO.1-shFOXA2 that expresses the shRNA of interest. After the transforming, restriction enzyme digestion and sequ encing were used to screen and verify the plasmids which were successfully con structed. The pLKO.1-shFOXA2 plasmid was then introduced to 293 T cells toget her with packaging plasmids psPAX2 and p MD2.G. The medium containing lenti virus was collected 48 h later. Then, the supernatant was centrifuged at 3000 rpm for 5 min at 4? to remove cell debris and passed through a 0.45 ?m filter. Th e control lentivirus lenti-shNC was obtained as well.3. The effect of FOXA2 on EMT in HCC cell linesReal-time PCR and Western blot were used to evaluate the expression of FOXA2 and EMT markers including E-cadherin and Vimentin in different HCC cell lines. After infected Focus with in different HCC cell lines, the expression of E-cadherin and Vimentin were detected and the morphologic change of Focus cells was observed. At the same time, the expression of E-cadherin and Vimentin in HepG2 cells was detected and morphologic change was observed after FOXA2 was knockdown.4. The role of FOXA2 in the migration and invasion abilities of HCC cell lines in vitroThe relationship between FOXA2 expression and migration abilities in different HCC cell lines was explored. With the up- and down-regulation methods, the changes of migration and invasion ability were tested by transwell assay.5. The effect of FOXA2 overexpression on the metastasis of HCC cell in vivoThe backbone plasmid pSin-EF1?-Luc for the production of lentivirus expressing Luciferase was constructed. After infected with lenti-Luc, puromycin screen, monoclone selection and luciferase expression test, a cell line Focus-Luc stablely expressing luciferase was constructed. 2 × 105 Focus-Luc cells infected with lenti-FOXA2 or lenti-GFP were inoculated subcutaneously into the left armpit of NOD/SCID mice. To ensure enough time for the tumor metastasis, mice were kept for 6 weeks until obvious tumors in the hypodermal tissue could be observed in both groups. For another model, 2 × 105 of Focus-Luc cells infected with lenti-FOXA2 or lentiGFP were injected through the tail vein(five in each group) and kept for 6 weeks. Mice were injected with 3 mg luciferin intraperitoneally under anesthesia and then organs including brain, lung, bone, liver, spleen and kidney were removed from the body quickly after the mice were killed. Metastasis nodules were monitored by the NightOWL LB 983 in vivo imaging system(Berthold Technologies).6. The effect of FOXA2 overexpression on MMP9 expression and the role of MMP9 in the FOXA2-induced inhibitory effect of anti-metastasis in HCCLentivirus was used to up-regulate FOXA2 in Focus cells and to down-regulated FOXA2 in HepG2 cells. Then, the expression level of MMP9 was tested by Real-time PCR and Western blot. To explore the potential mechanism of FOXA2-mediated regulation on MMP-9, we used JASPAR database to analyze the FOXA2-binding site in the region of the MMP-9 gene from-1000 to 3000 bp relative to the transcription start site. Six putative FOXA2-binding sites were predicted. The effect of FOXA2 on MMP-9 transcription was then examined with reporter assay using pGL3-promoter plasmids containing putative FOXA2-binding sites. The direct binding of FOXA2 on MMP9 gene was then proved by the Ch IP(Chromatin immunoprecipitation) assay. To determine the role of MMP-9 in the antimetastasis effect of FOXA2, Hep G2 cells were transfected with FOXA2 siRNA, MMP-9 siRNA or both constructs. The expression of FOXA2 and MMP-9 in the transfected cells was evaluated by real-time PCR the migration abilities of HepG2 in different group were accessed by transwell assay. To examine the effect of FOXA2 on MMP-9 expression in vivo, the mRNA and protein levels of FOXA2 and MMP-9 in subcutaneous tumor nodules in mice were determined. Moreover, the expression of FOXA2 and MMP9 in 72 human HCC specimens from was analyzed by real-time PCR and Western blot.7. Statistical analysisAll data are presented as mean ± SD. In this study, unpaired Student's t-test and ?2 test were applied for calculating statistical probability. A value of P < 0.05 was considered statistically signifiant and P < 0.01 was considered very signifiant. Statistical calculation was performed using the Statistical Program for Social Sciences software(SPSS, IBM). For all statistics, data from at least three independent samples or repeated experiments were used.?Results?1. The relationship between FOXA2 expression level and the progression of HCCThe expression of FOXA2 was decreased in 68.1%(49/72) of the HCC tissues relative to paired noncancerous adjacent tissues. The analysis of FOXA2 expression and clinicopathologic characteristics in HCC tissues indicated that the downregulation of FOXA2 correlated strongly with aggressive characteristics including venous invasion, poor differentiation and high TNM grade. Moreover, the mRNA level of FOXA2 was significantly lower in HCC with venous invasion group than that in non-venous invasion group. Consistently, western blot analysis showed the protein levels of FOXA2 were also decreased in HCC tissues with venous invasion. The decrease of FOXA2 expression in tumor tissues was even more obvious in the HCC-VI group(91.4%) than that in HCC group(46.0%) when compared with respective adjacent normal tissues(P < 0.001). FOXA2 expression level was even lower in PVTT tissues compared with primary tumor and noncancerous tissues.2. The construction of lenti-shFOXA2 and the control lentivirus lenti-sh NCThe backbone plasmids pSin-EF1?-FOXA2 and pLKO.1-shFOXA2 for producing overexpressing-lentivirus and knockdown lentivirus of FOXA2 were successfully constructed.3. The effect of FOXA2 on EMT in HCC cellsThe expression level of FOXA2 was correlated with E-cadherin in hepatoma cells. The level of FOXA2 was significantly higher in epithelial-like HCC cells, including HepG2 and Huh7, in comparison with that in mesenchymal-like HCC cells including MHCC-LM3 and Focus. As expected, overexpression of FOXA2 significantly increased E-cadherin level, inhibited Vimentin expression and resulted in striking morphological change in Focus cells. In contrast, the knockdown of FOXA2 promoted EMT in HepG2 cells.4. The effect of FOXA2 on the migration and invasion of HCC in vitroThe migratory ability of HCC cells with high FOXA2 expression levels was weaker than that with low FOXA2 expression. In Focus cells and MHCCLM3 cells, which both have strong metastasis capabilities, lenti-FOXA2 caused decreased migration and invasion compared with that of the control cells. In contrast, the knockdown of FOXA2 in HepG2 and Huh7, which have very weak metastatic capabilities, resulted in increased migration and invasion.5. The effect of FOXA2 on the metastasis of HCC in vivoTwo different HCC metastasis models were established in NOD/SCID mice. In the first model, metastasis nodules were evaluated after obvious subcutaneous tumours were formed. Even though the growth of nodules from Focus-Luc cells infected with FOXA2 lentivirus was slower than the control group, the mean volume of tumors in the two groups did not differ significantly at the endpoint(2.36 ± 2.40 cm3 vs. 3.90 ± 2.52 cm3, P = 0.403). However, overexpression of FOXA2 inhibited metastasis of Focus-Luc cells to bone and brain, as indicated by the luciferase signals. The bone and brain metastasis rate of the GFP group was 100%(5/5) and 20%(1/5), respectively, while that of FOXA2 group was 20%(1/5) and 0%(0/5), respectively. In the second model, Focus-Luc cells infected by injection through the tail vein with lenti-FOXA2 or lenti-GFP were forced to undergo metastasis. FOXA2 also inhibited Focus-Luc cells to form metastasis nodules in the lung. The lung metastasis rate of the GFP group was 60%(3/5), whereas no metastasis(0/5) was detected in the FOXA2 group.6. The effect of FOXA2 on the expression of MMP9 and thr role of MMP9 in the anti-metastasis effect of FOXA2 in HCCSix putative FOXA2 binding sites were predicted by using JASPAR database. Overexpression of FOXA2 reduced the luciferase activity of pGL3-B(1 – 988bp) plasmid in Focus cells while no effect of FOXA2 was found in the pGL3-A(1 – 511bp) plasmid. This approach identified a potential FOXA2 response region between 511 bp to 988 bp relative to the transcription start site of MMP-9 which is mostly located in the first intron(158 – 958bp). Ch IP assay confirmed the direct binding of FOXA2 in the first intron of MMP-9.?Conclusion?1. The downregulation of FOXA2 correlated with the progression of HCC.2. FOXA2 is a potent inhibitor of EMT in HCC cells, which suppresses the metastasis of HCC.3. MMP9 suppression is involved in the anti-metastasis effect of FOXA2.4. FOXA2 serve as a novel therapeutic target for the prevention of HCC metastasis clinically. |
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