Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model

Hepatitis B virus X antigen plays an important role in the development of human hepatocellular carcinoma (HCC). The key regulators controlling the temporal downstream gene expression for HCC progression remains unknown. In this study, we took advantage of systems biology approach and analyzed the microarray data of the HBx transgenic mouse as a screening process to identify the differentially expressed genes and applied the software Pathway Studio to identify potential pathways and regulators involved in HCC. Using subnetwork enrichment analysis, we identified five common regulator genes: EDN1, BMP7, BMP4, SPIB and SRC. Upregulation of the common regulators was validated in the other independent HBx transgenic mouse lines. Furthermore, we verified the correlation of their RNA expression levels by using the human HCC samples, and their protein levels by using the human liver disease tissue arrays. EDN1, bone morphogenetic protein (BMP) 4 and BMP7 were upregulated in cirrhosis, BMP4, BMP7 and SRC were further upregulated in hepatocellular or cholangiocellular carcinoma samples. The trend of increasing expression of the common regulators correlates well with the progression of human liver cancer. Overexpression of the common regulators increases the cell viability, promotes migration and invasiveness and enhances the colony formation ability in Hep3B cells. Our approach allows us to identify the critical genes in hepatocarcinogenesis in an HBx-induced mouse model. The validation of the gene expressions in the liver cancer of human patients and their cellular function assays suggests that the identified common regulators may serve as useful molecular targets for the early-stage diagnosis or therapy for HCC.     

Synergism between the hbx gene and aflatoxin B-1 in the development of murine liver-cancer

Infection by hepatitis B virus (HBV) and exposure to dietary aflatoxin B-1 (AFB(1)) have both been implicated by epidemiological studies to be important risk factors in the development of hepatocellular carcinoma (HCC). Our ability to derive transgenic mice which develop liver cancer as a consequence of the expression of a single gene from HBV, the HBx gene, provides an opportunity to use this animal model to test whether AFB(1) can induce p53 mutations, particularly at codon 249, which are frequently detected in HCC and, as a result, act synergistically with HBV to accelerate the manifestation of disease. While AFB(1) significantly shortened the latency of tumor development in the HBx transgenic mice, the tumors did not have p53 mutations. As in tumors from the untreated transgenic mice, the p53 tumor suppressor protein is found bound to the HBx protein and sequestered in the cytoplasmic compartment of the tumor cell. Despite the frequent involvement of ras mutations in mouse tumors, we also have not detected activation of the ms p21 protein in the tumors from the AFB(1)-treated mice. We conclude that although AFB(1) can act as a co-factor with HBx to induce HCC in mice, its mode of action in vivo remains obscure.     

Short chain fatty acids delay the development of hepatocellular carcinoma in HBx transgenic mice

Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The HBV encoded oncoprotein, HBx, alters the expression of host genes and the activity of multiple signal transduction pathways that contribute to the pathogenesis of HCC by multiple mechanisms independent of HBV replication. However, it is not clear which pathways are the most relevant therapeutic targets in hepatocarcinogenesis. Short chain fatty acids (SCFAs) have strong anti-inflammatory and anti-neoplastic properties, suggesting that they may block the progression of chronic liver disease (CLD) to HCC, thereby identifying the mechanisms relevant to HCC development. This hypothesis was tested in HBx transgenic (HBxTg) mice fed SCFAs. Groups of HBxTg mice were fed with SCFAs or vehicle from 6 to 9 months of age and then assessed for dysplasia, and from 9 to 12 months of age and then assessed for HCC. Livers from 12 month old mice were then analyzed for changes in gene expression by mass spectrometry-based proteomics. SCFA-fed mice had significantly fewer dysplastic and HCC nodules compared to controls at 9 and 12 months, respectively. Pathway analysis of SCFA-fed mice showed down-regulation of signaling pathways altered by HBx in human CLD and HCC, including those involved in inflammation, phosphatidylinositol 3-kinase, epidermal growth factor, and Ras. SCFA treatment promoted increased expression of the tumor suppressor, disabled homolog 2 (DAB2). DAB2 depresses Ras pathway activity, which is constitutively activated by HBx. SCFAs also reduced cell viability in HBx-transfected cell lines in a dose-dependent manner while the viability of primary human hepatocytes was unaffected. These unique findings demonstrate that SCFAs delay the pathogenesis of CLD and development of HCC, and provide insight into some of the underlying mechanisms that are relevant to pathogenesis in that they are responsive to treatment.     

Identification, using cDNA macroarray analysis, of distinct gene expression profiles associated with pathological and virological features of hepatocellular carcinoma

It is still unclear as to whether the gene expression profile in HCV- or HBV-related HCC exhibits a degree of specificity and whether the development of HCC in a context of cirrhosis influences this gene profile. To address these issues, the expression profiles of 15 cases of HCC were analysed using cDNA macroarray. A global analysis and hierarchical clustering, demonstrated the heterogeneity of HCC patterns, with a majority of down-regulated genes. Statistical analysis clearly showed a distinction between the gene expression profiles of HCV- and HBV-related HCC. HBV-associated HCC exhibited involvement of different cellular pathways, those controlling apoptosis, p53 signalling and G1/S transition. In HCV-related HCC we identified a more heterogenous pattern with an over-expression of the TGF-beta induced gene. In HCC developing on non-cirrhotic tissues, beta-catenin encoding gene and genes implicated in the PKC pathway were specifically up-regulated. In addition, our investigation highlighted a distinct profiles of TGF-beta superfamily encoding genes in well, moderately or poorly differentiated HCC. Overall, our study supports the hypothesis that despite the heterogeneity of the HCC pattern, the large-scale screening of gene expression may provide data significant to our understanding of the mechanism of liver carcinogenesis.     

Hepatitis B Virus X Protein Inhibits Tumor Suppressor miR-205 through Inducing Hypermethylation of miR-205 Promoter to Enhance Carcinogenesis

The infection of hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma (HCC), in which HBV X protein (HBx) plays crucial roles. MicroRNAs are involved in diverse biologic functions and in carcinogenesis by regulating gene expression. In the present study, we aim to investigate the underlying mechanism by which HBx enhances hepatocarcinogenesis. We found that miR-205 was downregulated in 33 clinical HCC tissues in comparison with adjacent noncancerous hepatic tissues. The expression levels of miR-205 were inversely correlated with those of HBx in abovementioned tissues. Then, we demonstrated that HBx was able to suppress miR-205 expression in hepatoma and liver cells. We validated that miR-205 directly targeted HBx mRNA. Ectopic expression of miR-205 downregulated HBx, whereas depletion of endogenous miR-205 upregulated HBx in hepatoma cells. Notably, our data revealed that HBx downregulated miR-205 through inducing hypermethylation of miR-205 promoter in the cells. In terms of function, the forced miR-205 expression remarkably inhibited the HBx-enhanced proliferation of hepatoma cells in vitro and in vivo, suggesting that miR-205 is a potential tumor-suppressive gene in HCC. HBx-transgenic mice showed that miR-205 was downregulated in the liver. Importantly, HBx was able to abrogate the effect of miR-205 on tumor suppression in carcinogenesis. Therefore, we conclude that HBx is able to inhibit tumor suppressor miR-205 to enhance hepatocarcinogenesis through inducing hypermethylation of miR-205 promoter during their interaction. Therapeutically, miR-205 may be useful in the treatment of HCC.     

Identification of genes differentially expressed in human hepatocellular carcinoma by a modified suppression subtractive hybridization method

To identify differentially expressed genes in human HCC in China, we applied a modified SSH method for cDNA subtraction. Such modification has made the method more effective for subtraction. We have obtained 36 and 24 differentially expressed cDNA fragments after modified SSH from 4 paired samples of human HCC and non-HCC tissues, respectively. Reverse Northern blotting analysis was performed to further identify the genes differentially expressed in the HCC and non-HCC tissue samples. There were 25 genes really overexpressed in HCC, and their corresponding encoding molecules may reflect the events of cell accelerated metabolism, proliferation, angiogenesis, anti-apoptosis, tumorigenesis (TLH107, TFH9) and the potential for metastasis. Of the 25 genes overexpressed in HCC, 5 were novel and their full-length cDNAs were cloned. These 5 novel genes are functionally associated with the occurrence and development of HCC according to the Database analysis. In the paired non-HCC tissues, there were 15 genes lowly or not expressed in HCC, and their encoding proteins function as tumor suppressors (TFA3, TFA11), acute-phase reactive proteins, and the blood plasma proteins that are mainly or exclusively synthesized in the liver. The distinct profiles of the differentially expressed genes in HCC and the paired non-HCC tissues have partially reflected the genetic alterations during HCC tumorigenesis. The novel HCC-specific gene TLH6 and the CT antigen encoding gene TLH107 may have diagnostic and therapeutic potentials in HCC and/or other solid cancers.     

Central genetic alterations common to all HCV-positive, HBV-positive and non-B, non-C hepatocellular carcinoma: a new approach to identify novel tumor markers

Hepatocellular carcinoma (HCC) is a common malignancy, but the prognosis remains poor due to the lack of sensitive diagnostic markers. To gain insight into the central molecular features common to all types of HCC, and to identify novel diagnostic markers or therapeutic targets for HCC, we performed a gene expression profiling analysis using a high throughput RT-PCR system. After examining the mRNA expression of 3,072 genes in 204 (119 tumor and 85 non-tumor) liver samples, we identified differential gene expression between the HCV group (n=80), HBV group (n=19) and non-B, non-C group (n=20) with a principal component analysis and a correlation spectrum analysis. After selection of genes differentially expressed between tumor and non-tumor tissues (p<0.01) within each HCC group, a total of 51 differentially expressed genes (23 upregulated and 28 downregulated genes) were found to be common to the three HCC groups. Gene Ontology grouping analysis revealed that genes with functions related to cell proliferation or differentiation and genes encoding extracellular proteins were found to be significantly enriched in these 51 common genes. Using an atelocollagen-based cell transfection array for functional analysis of eight upregulated genes, five (CANX, FAM34A, PVRL2, LAMR1, and GBA) significantly inhibited cellular apoptosis by two independent assays. In conclusion, we identified 51 differentially expressed genes, common to all HCC types. Among these genes, there was a high incidence of anti-apoptotic activity. This combination approach with the advanced statistical methods and the bioinformatical analysis may be useful for finding novel molecular targets for diagnosis and therapy.     

Identification of heterogeneous nuclear ribonucleoprotein as a candidate biomarker for diagnosis and prognosis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with a high mortality rate. However, spliceosomal genes are still lacking in the diagnosis and prognosis of HCC.MethodsIdentification of differentially expressed genes (DEGs) was performed using the limma package in R software. Modules highly related to HCC were obtained by weighted gene co-expression network analysis (WGCNA), and the module genes were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The biomarker for diagnosing HCC was determined by receiver operating characteristic (ROC) curve analysis, and the effect of the biomarker in the diagnosis of HCC was evaluated by performing five-fold cross-validation with logistic regression. HCC specimens from preoperatively treated patients were tested for biomarker by real-time quantitative polymerase chain reaction (RT-qPCR). Kaplan-Meier analysis was used to assess the relationship between biomarker and patient survival. The role of biomarker was evaluated using ESTIMATE analysis in the tumor microenvironment.ResultsIn this study, 389 DEGs were screened out from three Gene Expression Omnibus (GEO) datasets. We also found that the turquoise module of 123 genes from The Cancer Genome Atlas (TCGA) data was the key module with the highest correlation with HCC traits. Then, 123 genes were analyzed using the KEGG enrichment pathway, and eight genes were found to be most significantly related to the spliceosome pathway. We selected 8 genes and 389 DEGs shared genes, and finally got the only gene, heterogeneous nuclear ribonucleoprotein (hnRNPU). The high expression of hnRNPU was associated with poor prognosis of HCC, and hnRNPU was a biomarker for diagnosing HCC. In the tissues of patients with excellent HCC treatment hnRNPU messenger RNA (mRNA) was lower than in the tissues of patients with poor HCC treatment. High expression of hnRNPU was significantly increased in HCC patients with low stromal (P<0.05), low immune (P<0.05), and low estimation scores (P<0.05), and with high tumor purity (P<0.05) and high malignant progression (P<0.05) of the HCC.ConclusionsThe hnRNPU gene identified in this study may become a new biomarker for the diagnosis and prognosis of HCC.     

Chemopreventive effect of Curcuma longa Linn on liver pathology in HBx transgenic mice

Unlike other forms of hepatocellular carcinoma (HCC), HCC induced by hepatitis B virus (HBV) infection shows a poor prognosis after conventional therapies. HBV induces liver cirrhosis and HCC. Many researchers have made efforts to find new substances that suppress the activity of HBV. Curcuma longa Linn (CLL) has been used for traditional medicine and food in Asia, especially in India, and has shown chemopreventive effects in a HBV-related in vitro model. This in vivo study was designed to seek the chemopreventive effects of CLL and its mechanisms. CLL mixture concentrated with dextrose water by boiling was lyophilized. CLL extracts were administrated to HBV X protein (HBx) transgenic mice aged 4 weeks for 2 to 4 weeks and aged 6 months for 3 months. After administration, histological changes in the liver tissue and expression of HBx-related genes were investigated. CLL-treated mice showed less visceral fat, a smaller liver/body weight ratio and delayed liver pathogenesis. Proliferating cell nuclear antigen (PCNA) expression was also increased in CLL-treated HBx transgenic mice, indicating regeneration of damaged liver tissue. CLL treatment decreased expression of HBx and increased p21 and cyclin D1 in livers of HBx transgenic mice. In addition, p-p53 was increased after CLL treatment. These results suggest that CLL can have beneficial effects on the early and late stages of liver pathogenesis, preventing and delaying liver carcinogenesis. This drug should be considered as a potential chemopreventive agent for HBV-related hepatocarcinogenesis.     

Identification of transaldolase as a novel serum biomarker for hepatocellular carcinoma metastasis using xenografted mouse model and clinic samples

Hepatocellular carcinoma (HCC) is one of serious disorders with the highest morbidities and mortalities worldwide. Metastasis is the major concern that causes death in HCC. The goal of this study was to screen and identify potential serum proteins indicating HCC metastasis. Serum samples collected from control and HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1 wk, 3 wks and 6 wks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. A total of 554 proteins were identified and 80 proteins were differential expressed at least between one adjacent time points. Among them, expression level of transaldolase (TALDO) was validated in mouse and human serum. The level of TALDO protein was found to be higher in metastatic mice serum compared to that of non-metastatic mice. Human specific TALDO was then identified in mouse serum through human specific peptides. Immunohistochemical and western blot analysis showed that the expression of TALDO in human HCC tissues and HCC cell lines was associated with its metastatic behavior. Subsequent screening of TALDO expression in 72 clinical serum samples (comprising 36 non-metastatic HCC and 36 metastatic HCC samples) revealed higher TALDO level in the serum of metastatic HCC patients. A receiver operating characteristic (ROC) curve estimated a maximal sensitivity of 77.8% and 86.1% specificity for TALDO in detection of HCC metastasis. The present results demonstrated that the nude mouse xenograft model is an efficient system for performing metastasis-related biomarker discovery. TALDO may be useful biomarkers for the detection of HCC metastasis.     

Identification of Genes Associated with Dedifferentiation of Hepatocellular Carcinoma with Expression Profiling Analysis

To identify the genes associated with dedifferentiation of hepatocellular carcinoma (HCC), gene expression profiles of HCCs of well-and moderately differentiated grades were compared by means of oligonucleotide arrays. One tumor showed a nodule-in-nodule appearance (NIN), which is occasionally observed in the course of progression of HCC from well to less differentiated grade, when an inner, moderately differentiated tumor (MD) develops sequentially from the outer, well-differentiated tumor (WD). Seventy-six genes were identified to be up-regulated more than 3-fold and 33 genes were down-regulated in the inner nodule in NIN. By statistical analysis of the profiles from 10 individual additional liver tumors, 5 WDs and 5 MDs, we were able to identify 12 genes, LAMA3, PPIB, ADAR, PSMD4, NDUFS8, D9SVA, CCT3, GBAP, ARD1, RDBP, CSRP2 , and TLE1 , with significantly elevated expression, and 4 genes, CP, IL7R, CD48 , and PLGL , with decreased expression in MD. These selected genes were further validated using another 12 tumors, 5 WDs and 7 MDs, with semi-quantitative RT-PCR. We also applied neighborhood analysis to list the genes with high predictability values as most closely correlated with WD-MD distinction. Seven genes, ADAR, PSMD4, D9SVA, CCT3, GBAP, RDBP , and CSRP2 , whose expression was elevated and one gene, IL7R , whose expression was decreased, were included among the top 50 predictor genes. These genes are likely to be associated with dedifferentiation of HCC and their identification may help to elucidate the mechanism of liver cancer progression.     

Identification of genes associated with dedifferentiation of hepatocellular carcinoma with expression profiling analysis.

To identify the genes associated with dedifferentiation of hepatocellular carcinoma (HCC), gene expression profiles of HCCs of well-and moderately differentiated grades were compared by means of oligonucleotide arrays. One tumor showed a nodule-in-nodule appearance (NIN), which is occasionally observed in the course of progression of HCC from well to less differentiated grade, when an inner, moderately differentiated tumor (MD) develops sequentially from the outer, well-differentiated tumor (WD). Seventy-six genes were identified to be up-regulated more than 3-fold and 33 genes were down-regulated in the inner nodule in NIN. By statistical analysis of the profiles from 10 individual additional liver tumors, 5 WDs and 5 MDs, we were able to identify 12 genes, LAMA3, PPIB, ADAR, PSMD4, NDUFS8, D9SVA, CCT3, GBAP, ARD1, RDBP, CSRP2, and TLE1, with significantly elevated expression, and 4 genes, CP, IL7R, CD48, and PLGL, with decreased expression in MD. These selected genes were further validated using another 12 tumors, 5 WDs and 7 MDs, with semi-quantitative RT-PCR. We also applied neighborhood analysis to list the genes with high predictability values as most closely correlated with WD-MD distinction. Seven genes, ADAR, PSMD4, D9SVA, CCT3, GBAP, RDBP, and CSRP2, whose expression was elevated and one gene, IL7R, whose expression was decreased, were included among the top 50 predictor genes. These genes are likely to be associated with dedifferentiation of HCC and their identification may help to elucidate the mechanism of liver cancer progression.     

Hepatitis B virus X protein mutant upregulates CENP-A expression in hepatoma cells

The carcinogenic role of hepatitis B virus x protein (HBx) in hepatocellular carcinoma (HCC) remains largely unknown. Centromere protein A (CENP-A) has been found to be frequently overexpressed in HCC. In the present study, we aimed to investigate the role of HBx in regulating CENP-A activity in HCC carcinogenesis. CENP-A expression was examined and the HBx gene was sequenced in 20?HBsAg-positive HCC patients and corresponding non-cancerous liver tissues. The influence of HBx mutants on CENP-A expression in HepG2 cells was analyzed by a series of assays. We found that CENP-A expression was significantly elevated in HCC tissues. HBx deletion, especially the COOH-terminal deletion of HBx is a frequent event in HBV-associated HCC tissues. A positive correlation was found between CENP-A expression and HBx COOH mutation in HCC tissues. HBx mutant increased the expression of the CENP-A mRNA and protein compared with full-length HBx. However, HBx did not directly interact with CENP-A. It is concluded that overexpression of CENP-A is closely associated with HBx COOH mutation in HCC. HBx mutant can increase CENP-A expression, probably through a mechanism independent of their physical combination, and thereby it may represent a potential therapeutic strategy for HCC.     

用cDNA微阵列技术研究HBV X基因与AFB1对HBVx转基因小鼠药物代谢酶基因表达谱的影响

目的研究乙型肝炎病毒X基因和黄曲霉毒素诱发小鼠肝癌过程中药物代谢酶基因表达谱的变化,探讨两因素协同致肝癌的机制.方法用BiostarM-40s微阵列芯片比较研究HBVx组、AFB1组和(AFB1 HBVx)组的肝组织基因表达谱与对照组的差异.结果各实验组分别与对照组相比基因表达谱发生了明显的改变,各实验组上调与下调的基因数目分别为(AFB1 HBVx)组69项;AFB1组101项;HBVx组35项;其中与代谢酶相关的基因有18项表达发生改变,分别为(AFB1 HBVx)组13项(13/18,72%);HBVx组4项(4/18,22%);AFB1组8项(8/18,44%).结论小鼠受到HBV X基因和AFB1双重攻击后,其体内的GST、EPHX和UDPGT等药物代谢酶基因表达水平明显低于HBVx组和AFB1组.HBV与AFB1协同致癌的分子机制很可能与两者引起药物代谢酶基因表达水平下调有关.