Chao Ou

Proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) and functional identification of candidate protein peroxiredoxin II

In order to explore the proteins responsible for hepatocellular carcinoma (HCC), aflatoxin B1‐induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) was analyzed with 2‐DE and MS. By comparing HCC samples with their own precancerous biopsies and HCC‐surrounding tissues, a group of candidate proteins that differentially expressed in HCC were obtained. Peroxiredoxin (Prx) II, one of the candidates with distinct alteration, was further investigated and validated. Western blot and RT‐PCR assays confirmed the overexpression of Prx II in both tree shrew and human HCC tissues. RNA interference for silencing Prx II was employed subsequently to explore the function and underlying mechanism of Prx II on liver cancer cell line Hep3B. Results showed the cell proliferation and clone formation decreased obviously when Prx II expression was inhibited, while the flow cytometer analysis showed the percentage of cell apoptosis enhanced. Inhibition of Prx II expression also obviously increased the generation of ROS and malondialdehyde, both are the products from peroxidation. These results imply the important role of Prx II in hepatocarcinogenesis, possibly through its function in regulating peroxidation and hereby to provide a favorable microenvironment for cancer cell surviving and progressing.

Candidate genes responsible for human hepatocellular carcinoma identified from differentially expressed genes in hepatocarcinogenesis of the tree shrew (Tupaia belangeri chinesis)

Aim:  To explore gene expression profiles during hepatocarcinogenesis of the tree shrew, and to find the genes responsible for human hepatocellular carcinoma (HCC).

Histopathological changes in the liver of tree shrew (Tupaia belangeri chinensis) persistently infected with hepatitis B virus

BackgroundAn animal model for HBV that more closely approximates the disease in humans is needed. The tree shrew (Tupaia belangeri) is closely related to primates and susceptible to HBV. We previously established that neonatal tree shrews can be persistently infected with HBV in vivo, and here present a six year follow-up histopathological study of these animals.MethodsGroup A consists of six tree shrews with persistent HBV infection, group B consists of three tree shrews with suspected persistent HBV infection, while group C consists of four tree shrews free of HBV infection. Serum and liver tissues samples were collected periodically from all animals. HBV antigen and HBV antibodies were detected by ELISA and/or TRFIA. HBV DNA in serum and in liver biopsies was measured by FQ-PCR. Liver biopsies were applied for general histopathologic observation and scoring, immunohistochemical detections of HBsAg and HBcAg, and ultrastructural observation with electron microscope technique.ResultsHydropic, fatty and eosinophilic degeneration of hepatocytes, lymphocytic infiltration and hyperplasia of small bile ducts in the portal area were observed in group A. One animal infected with HBV for over six years showed multiple necrotic areas which had fused to form bridging necrosis and fibrosis, and megalocytosis. The hepatic histopathological scores of group A were higher than those of group B and C. The histopathological score correlated positively with the duration of infection.ConclusionsHepatic histopathological changes observed in chronically HBV-infected tree shrews are similar to those observed in HBV-infected humans. The tree shrew may represent a novel animal model for HBV infection.

Experimental chronic hepatitis B infection of neonatal tree shrews ( Tupaia belangeri chinensis ): A model to study molecular causes for susceptibility and disease progression to chronic hepatitis in humans

BackgroundHepatitis B virus (HBV) infection continues to be an escalating global health problem. Feasible and effective animal models for HBV infection are the prerequisite for developing novel therapies for this disease. The tree shrew (Tupaia) is a small animal species evolutionary closely related to humans, and thus is permissive to certain human viral pathogens. Whether tree shrews could be chronically infected with HBV in vivo has been controversial for decades. Most published research has been reported on adult tree shrews, and only small numbers of HBV infected newborn tree shrews had been observed over short time periods. We investigated susceptibility of newborn tree shrews to experimental HBV infection as well as viral clearance over a protracted time period.ResultsForty-six newborn tree shrews were inoculated with the sera from HBV-infected patients or tree shrews. Serum and liver samples of the inoculated animals were periodically collected and analyzed using fluorescence quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, Southern blot, and immunohistochemistry. Six tree shrews were confirmed and four were suspected as chronically HBV-infected for more than 48 (up to 228) weeks after inoculation, including three that had been inoculated with serum from a confirmed HBV-infected tree shrew.ConclusionsOutbred neonatal tree shrews can be long-term chronically infected with HBV at a frequency comparable to humans. The model resembles human disease where also a smaller proportion of infected individuals develop chronic HBV related disease. This model might enable genetic and immunologic investigations which would allow determination of underlying molecular causes favoring susceptibility for chronic HBV infection and disease establishment vs. viral clearance.

Effect of Ginkgo biloba extract on the expressions of Cox-2 and GST-Pi in rats with hepatocellular carcinoma risk

BACKGROUND Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers worldwide, and the pathogenesis is complicated at present. There iare few effective therapeutic measures, and novel therapeutic strategies are urgently required to improve clinical outcome. Ginkgo biloba extract (EGb) is reported to have an anti-cancer activity. OBJECTIVES To explore the effect of EGb on expressions of cyclooxygenase-2 (Cox-2) and glutathione S-transferase Pi (GST-Pi) in the pathogenesis of HCC. METHODS 120 Wistar rats were divided into three groups at random: normal control group (control group), HCC risk group without treatment (HCC risk group), HCC risk group treated with EGb (EGb group); n=40, respectively. The HCC risk in rat was induced by aflatoxin B1 injection. At the end of 13-week, 33-week, 53-week and 73-week, 10 rats in each group were killed and the relevant samples were collected. RESULTS The mRNA and protein expressions of Cox-2 and GST-Pi were measured by real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and western-blot. When compared with those in the control group in 73-week, the mRNA and protein expressions of GST-Pi in EGb group were weaker than those in HCC risk group in 73-week. However, the mRNA and protein expressions of Cox-2 in HCC risk group were increased than that of control group, and there was no statistical difference for mRNA and protein expressions of Cox-2 between HCC risk group and EGb group. CONCLUSION EGb can regulate the expression of GST-Pi, but it does not seem to have an effect on Cox-2 expression in the liver of HCC risk rats.

Progress on establishment of tree shrew (Tupaia) chronic infection with HBV in vivo

Hepatitis B virus (HBV) infection is one of the important health problems worldwide, especially in China. Feasible and effective animal models of HBV infection in vivo are prerequisite for the HBV-related basic and clinical studies. Located in the highly prevalent region of HBV and hepatocellular carcinoma (HCC), the laboratory of Guangxi Cancer Institute has focused on the cause, pathogenesis and chemoprevention of HCC, and has started the work of establishing tree shrew (Tupaia) models of HBV infection in vivo since the early 1980s. This paper provides an overview of the research process, and highlights the new progress on the chronic infection of tree shrews after inoculated with HBV neonatally in vivo.

Identification of key genes in hepatocellular carcinoma and validation of the candidate gene, cdc25a, using gene set enrichment analysis, meta-analysis and cross-species comparison.

The aim of the present study was to determine key pathways and genes involved in the pathogenesis of hepatocellular carcinoma (HCC) through bioinformatic analyses of HCC microarray data based on cross-species comparison. Microarray data of gene expression in HCC in different species were analyzed using gene set enrichment analysis (GSEA) and meta-analysis. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to determine the mRNA and protein expression levels of cdc25a, one of the identified candidate genes, in human, rat and tree shrew samples. The cell cycle pathway had the largest overlap between the GSEA and meta-analysis. Meta-analyses showed that 25 genes, including cdc25a, in the cell cycle pathway were differentially expressed. Cdc25a mRNA levels in HCC tissues were higher than those in normal liver tissues in humans, rats and tree shrews, and the expression level of cdc25a in HCC tissues was higher than in corresponding paraneoplastic tissues in humans and rats. In human HCC tissues, the cdc25a mRNA level was significantly correlated with clinical stage, portal vein tumor thrombosis and extrahepatic metastasis. Western blotting showed that, cdc25a protein levels were significantly upregulated in HCC tissues in humans, rats and tree shrews. In conclusion, GSEA and meta-analysis can be combined to identify key molecules and pathways involved in HCC. This study demonstrated that the cell cycle pathway and the cdc25a gene may be crucial in the pathogenesis and progression of HCC.