Expression of epidermal fatty acid-binding protein in cross-species hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the utility of the cross-species screening strategy for investigating key molecule(s) involved in onset and progression of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>HCC-related molecule data from our previous studies and in the literature were collected to establish a cross-species dataset. Tissue samples of HCC, non-HCC surrounding liver (para-HCC), and normal liver that were collected from humans, tree shrews and rats. The genes reported to have the most differential expression in HCC were verified by analyzing the mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively.</p><p><b>RESULTS</b>The cross-species dataset of HCC-related molecules included four genes: epidermal fatty acid-binding protein (E-FABP), liver (L)-FABP, tyrosine a-ketoglutarate transaminase (TKT), and cytokeratin (CK8). In humans, E-FABP mRNA expression was significantly higher (P less than 0.05) in HCC (0.87+/-0.14 vs. para-HCC: 0.64+/-0.12 and normal liver: 0.67+/-0.07; F=20.910). Similar results were obtained in tree shrew (HCC: 0.87 +/- 0.25 vs. para-HCC: 0.73 +/- 0.19 and normal liver: 0.68+/-0.19; F=3.807) and rat (HCC: 0.97+/-0.22 vs. para-HCC: 0.78+/-0.16 and normal liver: 0.80 +/- 0.13; F=4.482). The Western blotting analyses revealed a similar statistically significant trend.</p><p><b>CONCLUSION</b>The cross-species screening strategy for tumor genes may represent a feasible and convenient process of identifying key molecule(s) for human HCC. E-FABP may be a particularly crucial molecule for hepatocarcinogenesis.</p>

Factors influencing long-term hepatitis B virus infection of the tree shrew (Tupaia belangeri chinensis) as an in vivo model of chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine the methods for establishing an in vivo model of long-term hepatitis B virus (HBV) infection in the Chinese tree shrew (Tupaia belangeri chinensis).</p><p><b>METHODS</b>Seventy-seven neonate (1-3 days old) and 49 young adult (2 weeks to 1 year old) tree shrews were inoculated with different HBV sources (chronic hepatitis B (CHB) human patient serum, single or pooled; HBV-infected tree shrew serum, single only; HBV-infected HepG2.2.15 cells' culture medium supernatant; HBV genome-transfected HepG2.2.15 cells' culture medium supernatant) through various routes of injection (subcutaneous, intraperitoneal, and direct liver via abdominal skin; adults also received intravenous and indirect liver via spleen). Serum and liver biopsies were collected from the animals at various time points post-inoculation for detection of HBV markers by fluorescence quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, time-resolved immunofluorescence, Southern blotting, dot blotting, immunohistochemistry, and microscopy.</p><p><b>RESULTS</b>Among the neonatal group of tree shrews, six (7.8%) were confirmed as HBV-infected for more than 72 (up to 228) weeks after inoculation and another seven (9.1%) were suspected of persistent infections. None of the young adult tree shrews developed persistent infection. Inoculation with single-source serum from either CHB humans or tree shrews were responsible for the most cases of infections, and the subcutaneous injection produced more infections than the other inoculation routes. The most reliable methods of determining HBV infection status were detection of serum HBV immunoreactive markers and intrahepatic HBV DNA.</p><p><b>CONCLUSION</b>In order to establish an in vivo model of CHB in the tree shrew, the animals should be inoculated in the neonatal period using subcutaneous injection.</p>

Effects of AKR1B10 gene silence on the growth and gene expression of HCC cell line MHCC97H / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the biological function and possible underlying mechanism of aldo-keto reductase family 1 member B10 (AKR1B10) gene during hepatocarcinogenesis.</p><p><b>METHODS</b>A pair of chemically synthesized small interfering RNA (siRNA) targeting on AKR1B10 was transfected into liver cancer cell line MHCC97H by LipofectamineTM 2000. After confirming the interfering effects of AKR1B10-siRNAs through Quant SYBR Green polymerase chain reaction (Real-time PCR), Western blot and enzymatic activity assay, the capabilities of proliferation and apoptosis of the transfected cells were observed by CCK-8 assay and flow cytometry analysis, and the expressions of a group of tumor-related gene such as c-myc, c-fos, N-ras were observed through Real-time PCR.</p><p><b>RESULTS</b>The expressions of AKR1B10 and the enzymatic activity were down-regulated significantly in AKR1B10-siRNA-transfected cells. Compared with mock and blank control groups, cell growth in AKR1B10-siRNA-transfected group was inhibited by 26.6%+/-3.1% at 72h after transfection. The ratio of apoptotic cells was 37.3%+/-1.0% in AKR1B10-siRNA-transfected group, which was significantly higher than that in mock and blank control groups (P < 0.01). Real-time PCR showed that the expressions of oncogene c-myc, c-fos and N-ras, and the proliferation-associated gene ki-67 were down-regulated in AKR1B10-siRNA-transfected cells, while the expressions of apoptosis-promoting gene caspas-3 and bax were up-regulated.</p><p><b>CONCLUSIONS</b>AKR1B10 might promote proliferation, inhibit apoptosis and then induce malignant transformation of hepatocytes by regulating the expression level of some tumor-related genes.</p>

Long-term observation of hepatitis B virus (HBV) replication in new-born tree shrews inoculated with HBV / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the hepatitis B virus (HBV) replication in the tree shrews that were inoculated with HBV at neonatal period.</p><p><b>METHODS</b>Six new-born tree shrews were inoculated with human HBV positive serum. Blood samples and liver biopsies were collected at different time points after inoculation. The HBV infection markers were tested by nested polymerase chain reaction (nPCR), fluorescence quantitative polymerase chain reaction (FQ-PCR), Southern blot, ELISA and immunohistochemistry staining. The liver tissues were observed under electron and light microscope.</p><p><b>RESULTS</b>48 weeks after inoculation, HBV DNA and HBV cccDNA were detected in the serum and liver samples of three animals (number 1, 2 and 6) by nPCR. The copy-numbers of HBV DNA detected by FQ-PCR in their serum and liver samples were 103 and-104/ml respectively,and the total DNA in 1microg liver tissue was 107-108. Southern blot indicated that HBV replication intermediates such as HBV cccDNA and HBV ssDNA was detectable in liver tissues. HBsAg was detected by ELISA, and immunohistochemical staining showed a gradual increase of HBsAg-positive liver cells. High copy number of HBV DNA and suspected HBV EM particles could be detected in the liver samples from one of the three animals that have survived more than 2 years after inoculation. The other three animals showed low HBV DNA copy number, and the rest of the signs of HBV infection were negative or transiently positive.</p><p><b>CONCLUSIONS</b>Neonatal tree shrews can be infected with human HBV. HBV can replicate inside the liver cells of tree shrew.</p>

Differentially expressed proteins in the precancerous stage of rat hepatocarcinogenesis induced by diethylnitrosamine / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen the differentially expressed proteins especially at the precancerous stage of diethylnitrosamine (DEN) induced hepatocarcinogenesis by comparative proteome research.</p><p><b>METHODS</b>Rats were divided into normal and DEN groups and sacrificed periodically. The liver samples were stained with gamma-glutamyl transpeptidase (GGT) and HE to distinguish the preneoplastic lesion (pre-HCC) from the normal and HCC tissues. The two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) were then applied to analyze the differentially expressed protein between pre-HCC and normal tissues, pre-HCC and HCC, as well as HCC and normal tissues. A few of the candidate proteins such as laminin receptor 1 (67LR) and agmatinase were validated by Western blot and RT-PCR.</p><p><b>RESULTS</b>Totally, there were 82 proteins that differentially expressed two fold or more in one kind of tissues sample than the other, 47 of which occurred in the pre-HCC tissues. Eight proteins including 67LR were consistently up-regulated from normal tissue to pre-HCC and then to HCC tissues, while 22 proteins including agmatinase showed progressively down-regulated in these tissues samples.</p><p><b>CONCLUSION</b>The protein expression profiles are different during the process of hepatocarcinogenesis. Further study on the differentially expressed protein, especially these upregulated in the precancerous stage such as 67LR and agmatinase, might contribute to prevention and early diagnosis of human HCC.</p>

The biological function of peroxiredoxin II on Hep3B cells and its underlying mechanism / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the biological function and its possible underlying mechanism of peroxiredoxin II (PrxII) in liver cancer cell line Hep3B.</p><p><b>METHODS</b>Two pairs of double-stranded small interfering RNA (siRNA) targeted on PrxII gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive oxygen species (ROS) and malondialdehyde (MDA), were applied to explore whether the antioxidant mechanism was involved in the effects of PrxII functioning on Hep3B cell.</p><p><b>RESULTS</b>The two pairs of siRNA significantly inhibited PrxII mRNA and protein expression. Compared to the mock and blank control groups, the two PrxII-silent groups showed decreased rates of cell growth and clone formation and increased rates of cell apoptosis. The numbers of the formed colonies were 42.0+/-2.8 and 40.5+/-0.7 respectively in the two PrxII-silent groups, while they were 121.5+/-2.1 and 130.0+/-1.4 in the mock and blank control groups (P less than 0.05). The levels of endogenous ROS and MDA were significantly higher in the two PrxII-silent groups than those in the mock and blank control groups (P less than 0.05).</p><p><b>CONCLUSION</b>PrxII might play an important role in the hepatocarcinogenesis, possibly through an antioxidant function which may provide a favorable microenvironment for cancer cell survival and progression.</p>

The expression of peroxiredoxin II in hepatocellular carcinoma and its significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the mRNA and protein expressions of peroxiredoxin II (PrxII) in hepatocellular carcinoma (HCC) and their significance.</p><p><b>METHODS</b>HCC was induced by aflatoxin B1 (AFB1) in 6 tree shrews (Tupaia belangeri chinensis). The expression levels of PrxII mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot on HCC tissues and on their surrounding liver tissues (para-HCC). Biopsied liver tissues were taken before the HCC induction (pre-HCC) from the same animals and from a group of blank controlled animals that served as controls. Liver biopsy specimens from 18 cases of human HCC and from 17 healthy human volunteers were studied using the same methods.</p><p><b>RESULTS</b>The mRNA and protein expressions of PrxII in tree shrew HCC tissues were significantly higher than those in para-HCC and pre-HCC tissues, and also higher than those in the liver tissues from the control animals (all P < 0.05). The expression levels of PrxII mRNA and protein in human HCC tissues were also significantly higher than those in their para-HCC tissues and in the human normal liver tissues (P < 0.05).</p><p><b>CONCLUSION</b>PrxII might play an important role in hepatocarcinogenesis and might be used as a molecular target for HCC prevention and treatment.</p>

Dynamic expression of survivin during hepatocarcinogenesis in rats / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine the dynamic expression of survivin gene in hepatocarcinogenesis of rats induced by aflatoxin B1 (AFB1).</p><p><b>METHODS</b>78 Sprague-Dawley rats were used in this study. Hepatocellular carcinoma was induced in the rats by aflatoxin B1. Liver and HCC tissues were examined by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The earliest hepatocellular carcinoma occurred at 46th week after AFB1 treatment. The HCC incidence was 54.9% (28/51) at 46th week and 64.9% (24/37) at 58th week. The positive rates of survivin protein expression in 24 HCC, para-cancerous liver tissues of experimental group were 41.7% and 54.2%, respectively, with no significant difference between them (P > 0.05). No survivin expression was detected in the experimental group before 46th week, neither in the rats without HCC occurrence nor the normal controls. The level of survivin mRNA expression in HCC at 58th week was significantly higher than that in pre-HCC, no-HCC and normal liver tissues in the control group (P < 0.01). The level of survivin mRNA expression in para-carcinoma tissues was also significantly higher than that in no-HCC and normal liver tissues of the control (P < 0.01). The level of survivin mRNA in pre-HCC at 12th, 20th, 36th, 46th weeks were significantly higher than those in normal liver tissues taken from control group during the same periods (P < 0.01).</p><p><b>CONCLUSION</b>The over-expression of survivin gene is related to the occurrence of HCC and may play an important role in the carcinogenesis of HCC.</p>

A cDNA microarray study of the differential expression of genes in signal transduction pathway during hepatocarcinogenesis in tree shrews / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the differential expression of genes in signal transduction pathway (STP) during the hepatocarcinogenesis in tree shrews induced by AFB1 and/or HBV and to elucidate the molecular mechanism of hepatocellular carcinoma (HCC) development.</p><p><b>METHODS</b>Adult tree shrews were divided into three groups: Group A was fed AFB1 only, Group B was infected firstly with HBV then fed AFB1 as in Group A, Group C served as the normal control. Liver biopsies were obtained at the 30th, 60th and 90th week of the experiment or until HCC occurred and the animals were sacrificed. Tree shrew-specific cDNA microarray was applied for detecting the differential expression of corresponding genes in each group at different time points during the experiment, and real time RT PCR was applied to verify the results of the cDNA microarray.</p><p><b>RESULTS</b>Genes of IGF-II, C-rel, and NF-kappaB2 were differentially expressed between para-cancerous tissues and HCC tissues in both group A and group B, and the differential expression of bcl-2, cyclin A and CNTF was only seen in group B. Between the experimental groups A and B and the control group C, there were differential expressions of CNTF and cyclin A in the early 30th week and middle 60th week stage of hepatocarcinogenesis in tree shrews. Real time RT PCR results showed that the expression level of IGF-II and C-Rel in group A and of IGF-II in group B in HCC tissues were significantly lower than that in the adjacent non-cancerous tissues and in the biopsies taken at the 30th and 60th week of the experiment. Nevertheless, there were no significant differences between the para-cancerous tissues and the cancer tissues at the 30th and 60th week. These results were consistent with the cDNA microarray assay. The expression levels of C-Rel and CNTF in group B were not obviously altered in the para-cancerous tissues, HCC and at the 60th week, but they were significantly lower in these tissues than that in the tissues at the 30th week. In group A, the expression levels of CNTF in adjacent liver and HCC tissues were higher than that in para-cancerous lesions, but the difference did not reach a statistically significant level. In group C, the expression level of IGF-II, C-Rel and CNTF at different stages showed no significant differences, which was consistent with the cDNA microarray results.</p><p><b>CONCLUSIONS</b>To apply the tree shrew-specific cDNA microarray to detect the differential expression of genes related to signal transduction pathway during tree shrew hepatocarcinogenesis could be a valuable utility for further comprehending the mechanism of HCC. IGF-II, NF-kappaB2, C-rel, Bcl-2, and cyclin A. CNTF may be involved in the occurrence and progress of HCC in tree shrews.</p>

Different gene expression during hepatocarcinogenesis in tree shrew induced by aflatoxin B1 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To understand the molecular mechanism and find out the responsible genes for liver cancer by exploring the regulation of gene expression during hepatocarcinogenesis in tree shrew induced by aflatoxin B1 (AFB1).</p><p><b>METHODS</b>The tissues from tree shrew of different stages during the pathogenesis and development of hepatocellular carcinoma (HCC), liver cancer tissue, para-cancerous tissues, pre-cancerous liver tissues, liver tissues of the same stage from normal controls and the liver tissues taken before AFB1-treatment were analyzed for gene expression by cDNA array.</p><p><b>RESULTS</b>Four patterns of gene expression were observed during AFB1-induced hepatocarcinogenesis. They were: genes up-regulated in HCC tissue and para-cancerous tissue, especially in HCC tissues; genes with similar expressing level in both HCC tissue and para-cancerous tissue, but higher than that in pre-cancerous tissue; genes down-regulated in HCC tissue; genes up-regulated before HCC appeared but down-regulated after HCC appeared.</p><p><b>CONCLUSION</b>Dynamic observation of gene expression will be beneficial to elucidate the mechanisms of AFB1- induced hepatocarcinogenesis and locate the responsible genes.</p>

Expression of tumor-specific cancer/testis antigens in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>(1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression.</p><p><b>METHODS</b>The cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes.</p><p><b>RESULTS</b>The MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05).</p><p><b>CONCLUSIONS</b>With a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.</p>

Alteration of p53 gene during tree shrews' hepatocarcinogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To detect the expression and variation of p53 gene during tree shrews' hepatocarcinogenesis induced by hepatitis B virus (HBV) and aflatoxin B1 (AFB1).</p><p><b>METHODS</b>Tree shrews were divided into four groups: the tree shrews were infected with HBV and fed with AFB1 in group A, only infected with HBV in group B, fed with AFB1 alone in group C, and normal control in group D. All the tree shrews were performed liver biopsy every 15 weeks. The tissues of liver and tumor were detected by immunohistochemistry and molecular biotechnologies.</p><p><b>RESULTS</b>(1) The incidence of hepatocellular carcinoma (HCC) in group A (66.7%) was higher than that in Group B and C (30%). HCC appearance in group A was earlier than that in group C (120.0 weeks +/-16.6 weeks vs 153.3 weeks +/-5.8 weeks, t = 3.336, P<0.01). (2) Mutated p53 protein was not found before the 75th week of the experiment in each group. (3) At the 105th week, the expression rates of mutated p53 protein were 78.6%, 60% and 71.4% in group A, B and C respectively, which were much higher than that (10%) in group D (x2 > or = 5.03, P<0.05). An abnormal band of p53 gene was detected in both group A and C. (4) The mutation points of p53 gene in liver cancer of tree shrew were at codon 275, 78 and 13. The nucleotide sequence and amino acids sequence of tree shrew's wild-type p53 showed 91.7% and 93.4% homology with those of human p53 respectively.</p><p><b>CONCLUSIONS</b>There is a remarkable synergistic effect between HBV and AFB1 on HCC. Mutated p53 protein is expressed before HCC occurrence, which promotes the development and progress of HCC. HBV and AFB1 may synergistically induce p53 gene mutation.</p>

Serial pathologic changes in livers of Tree shrews and Macaca assamensises infected with human Hepatitis B virus / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To serially observe the pathologic changes in livers of tree shrews and macaca assamensises infected with HHBV.</p><p><b>METHODS</b>10 adult tree shrews and 28 macaca assamensises were inoculated with HBV rich human sera. The liver of the animals were regularly biopsied. The liver samples were examined histopathologically by HE staining. Some samples were stained for HBsAg by immunohistochemistry (IH), and HBV DNA by in situ hybridization (ISH).</p><p><b>RESULTS</b>HBsAg in 80% of tree shrews infected with HHBV can be detected by IH, HBV DNA in 50% of those can be found by ISH.The positive rates of HBsAg in macaca assamensises' livers were 25% by IH, none HBV DNA was detected.</p><p><b>CONCLUSION</b>The tree shrew model seems to be applicable for the research of human hepatitis B.</p>

Human hepatitis B virus infection of tree shrews and Macaca assamensis in vivo / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To examine sensitivity of the tree shrews and Macaca assamensis to human hepatitis B virus (HHBV) by serologic methods.</p><p><b>METHODS</b>Totally 233 tree shrews and 28 Macaca assamensis were inoculated with human sera containing HBV. After inoculation, the sera were collected weekly from them and HBV markers were detected with HBV ditecting ELISA kits.</p><p><b>RESULTS</b>Ninety percent of the tree shrews developed acute infection, among them, 44.4 % persisted for over one year, 33.3% of them developed chronic infection persisted for 2 years and one month; the persistence of HBV in Macaca assamensis was much shorter.</p><p><b>CONCLUSION</b>These data clearly indicated that tree shrew may be used as an animal model for study of chronic HBV infection, whereas, Macaca assamensis, showed only a transient sensitivity to HHBV.</p>