Gradual upregulation of OCI-5 expression during occurrence and progression of rat hepatocellular carcinoma

BACKGROUND: OCI-5, the rat homologene of human glypican 3 ( GPC3), is confirmed upregulated in hepatocellular carcinoma (HCC). The present study was undertaken to detect gene expression change of OCI-5 during occurrence and progression of rat HCC. METHODS: Male Sprague-Dawley rats were given diethyl-nitrosamine ( DENA) to induce HCC. Three DENA-induced rats and one control rat were sacrificed every week for 18 weeks during the development of HCC. Tissues specimens were snap-frozen in liquid nitrogen and total RNA was isolated. Sk-Hepl cells were treated with DENA at different concentrations. The gene expression levels of OCI-5 and GPC3 were detected with the RT-PCR method. RESULTS: OCI-5 was not expressed in normal rat liver tissues. When HCC occurred and aggravated, OCI-5 expression was gradually elevated to a very high level. GPC3 was not expressed in the DENA-treated Sk-Hepl cells. CONCLUSIONS: OCI-5 was not expressed in normal rat liver tissues but in rat HCC tissues. High-expression of OCI-5 in DENA-induced rat HCC model was the gene expression change of HCC not the DENA-induced gene expression. The expression level of OCI-5 was not only elevated in rat HCC but also gradually along the occurrence and progression of HCC, indicating that GPC3 might serve as a sensitive marker of early stage HCC.     

Differential expression of genes during aflatoxin B(1)-induced hepatocarcinogenesis in tree shrews

AIM:Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1),to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism.METHODS:Tree shrews ( Tupaia belangeri chinensis)were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding noncancerous liver tissues, 2 biopsied tissues at the early stage(30^th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0^th week) of the experiment, and another i mixed sample of two liver tissues from the untreated control animals biopsied at the 90^th week of the experiment. The samples were then tested with the method of Atlas^TM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared.RESULTS:The profiles of differently expressed genes were quite different in different ways of comparison.At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up-or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as “important genes” because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC,genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC.CONCLUSION: A considerable number of genes could change their expressing levels both in HCC and in HCC-surrounding non-cancerous liver tissues. A few modular genes were up-regulated only in HCC but not in surrounding liver tissues, while some apoptosis-related genes were down-regulated in HCC and up-regulated in surrounding liver tissues. To compare gene-expressing levels among the liver tissues taken at different time points during hepatocarcinogenesis may be helpful to locate the responsible gene (s) and understand the mechanism for AFB1 induced liver cancer.     

Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/β-catenin signalling pathway

AIM To reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride(CCl4).METHODS Eight-week-old male Wistar rats were randomly divided into a normal control group(C group), a CCl4 group(CCl4 group), a vitamin D group(V group), a puerarin group(P group), and a combined group of vitamin D and puerarin(V + P group), each of which contained ten rats. In this way, we built a rat model of CCl4-induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid(HA). We also measured hydroxyproline(Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the proteinlevels of Collagen I, Collagen III, Wnt1, and β-catenin in the liver tissues, respectively.RESULTS Hepatic fibrosis was observed in the CCl4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and β-catenin in liver were also decreased, as well as the protein levels of Wnt1 and β-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis.CONCLUSION The combined application of vitamin D and puerarin is capable of alleviating CCl4-induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/β-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect.     

Changes of ECM and CAM gene expression profile in the cirrhotic liver after HCV infection： Analysis by cDNA expression array

AIM: We aimed to observe the expression of extracellular matrix (ECM) and cellular adhesion molecules (CAM) in cirrhotic liver tissues after hepatitis C virus (HCV) infection. METHODS: Twelve patients with post HCV inflammatory liver cirrhosis were selected to evaluate their liver function and other virological,pathological parameters.Then three specimens of cirrhotic patients whose health assessment results and laboratory data were similar and three normal liver specimens explanted from liver grafts prepared for liver transplantation were chosen for investigating gene expression of ECM and CAM using cDNA expression array. RESULTS: The cDNA array assay revealed 36.7% (36/96) of genes with changes, in which 26.3% (26/96) was up-regulated and 10.1% (10/96) was down-regulated. Integrin (ITGA), collagen (COL), ADAMTS were identified as the characteristic changes of ECM and CAM gene expression levels. ITGA were demonstrated β1 and β2 sub-section changed in liver cirrhosis. CONCLUSION: ECM and CAM play an important role in the progression of liver cirrhosis after HCV infection. The capital mechanism is related to the inflammatory cells infiltration, the activation and transformation of ECM producing cells and the imbalance between production and elimination of ECM.     

A novel mouse model for colitis-associated colon carcinogenesis induced by 1,2-dimethylhydrazine and dextran sulfate sodium

AIM: To develop an efficient animal colitis-associated carcinogenesis model and to detect the expression of β-catenin and p53 in this new model. METHODS: Dysplasia and cancer were investigated in mice pretreated with a single intraperitoneal injection of 20 mg/kg body mass of 1,2-dimethylhydrazine prior to three repetitive oral administrations of 30 g/L dextran sulfate sodium to give conditions similar to the clinically observed active and remission phases. Immunohistochemical staining of β-catenin and p53 was performed on paraffin-imbedded specimens of animals with cancer and/or dysplasia, those without dysplasia and the normal control animals. RESULTS: At wk 11, four early-invasive adenocarcinomas and 36 dysplasia were found in 10 (90.9%) of the 11 mice that underwent 1,2-dimethylhydrazine-pretreatment with 3 cycles of 30 g/L dextran sulfate sodium-exposure. Dysplasia and/or cancer occurred as flat lesions or as dysplasia-associated lesion or mass (DALM) as observed in humans. Colorectal carcinogenesis occurred primarily on the distal portion of the large intestine. No dysplasia and/or cancer lesion was observed in the control groups with 1,2-dimethylhydrazine pretreatment or 3 cycles of 30 g/L dextran sulfate sodium exposure alone. Immunohistochemical investigation revealed that β-catenin was translocated from cell membrane to cytoplasm and/or nucleus in 100% of cases with dysplasia and neoplasm, while normal membrane staining was observed in cases without dysplasia and the normal control animals. Nuclear expression of p53 was not detected in specimens. CONCLUSION: A single dose of procarcinogen followed by induction of chronic ulcerative colitis results in a high incidence of colorectal dysplasia and cancer. Abnormalexpression of β-catenin occurs frequently in dysplasia and cancer. This novel mouse model may provide an excellent vehicle for studying colitis-related colon carcinogenesis.     

Ductular proliferation in liver tissues with severe chronic hepatitis B： An immunohistochemical study

AIM: To clarify the pathogenesis of ductular proliferation and its possible association with oval cell activation and hepatocyte regeneration. METHODS: Immunohistochemical staining and image analysis of the ductular structures in the liver tissues from 11 patients with severe chronic hepatitis B and 2 healthy individuals were performed. The liver specimens were sectioned serially, and then cytokeratin 8 (CK8), CK19, OV6, proliferating cell nuclear antigens (PCNA), glutathione-S-transferase (GST),α-fetal protein (AFP) and albumin were stained immunohistochemically. RESULTS: Typical and atypical types of ductular proliferation were observed in the portal tracts of the liver tissues in all 11 patients. The proliferating ductular cells were positive for CK8, CK19, OV6 and PCNA staining. Some atypical ductular cells displayed the morphological and immunohistochemical characteristics of hepatic oval cells. Some small hepatocyte-like cells were between hepatic oval cells and mature hepatocytes morphometri-cally and immunohistochemically. CONCLUSION: The proliferating ductules in the liver of patients with severe chronic liver disease may have different origins. Some atypical ductular cells are actually activated hepatic oval cells. Atypical ductular proliferation is related to hepatocyte regeneration and small hepatocyte-like cells may be intermediate transient cells between hepatic oval cells and mature hepatocytes.     

Expression of transforming growth factor-α and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

AIM:To evaluate the expression of transforming growthfactor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg)in human hepatocellular carcinoma (HCC) tissues and itssignificance.METHODS:Seventy specimens of HCC tissues weredetected by immunohistochemical method.Five specimensof normal human liver tissues were used as control.RESULTS:The TGF-α positive expression rates in HCCand its surrounding tissues were 74.3%(52/70) and88.1%(52/59),respectively.TGF-α positive granules weremainly in the cytoplasm and fewer existed on thekaryotheca.The TGF-α positive expressing rate in welldifferentiated HCC was significantly higher than that inmoderately and poorly differentiated HCC (P<0.05).TheTGF-α positive expression also was observed in intrahepaticbile ducts (part of those were hyperplastic ducts).TheHBsAg positive expression rates in HCC and its surroundingtissues were 21.4%(15/70) and 79.7%(47/59),respectively.HBsAg positive granules were in the cytoplasm,inclusionand on the karyotheca.There was a prominent positivecorrelation between TGF-a and HBsAg expression in HCCsurrounding tissues (P<0.05,γ=0.34).TGF-α was usuallyexisted with HBsAg in regenerated and/or dysplastic livercells.In the five normal liver tissues,TGF-α and HBsAgwere not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is closely relatedwith hepatocarcinogenesis.The overexpression of TGF-αin the liver seems to be associated with the regenerationof hepatocytes injured by HBsAg.The continued expressionof TGF-α might lead to dysplasia of liver cells anddevelopment of HCC.Furthermore,TGF-α might play arole in morphogenesis and regeneration of intrahepaticbile ducts.     

ShRNA-mediated gene silencing of β-catenin inhibits growt of human colon cancer cells

AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205. METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry. RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level. The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing. CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.     

Differential expression of genes during aflatoxin B_1-induced hepatocarcinogenesis in tree shrews

AIM:Through exploring the regulation of gene expressionduring hepatocarcinogenesis induced by aflatoxin B_1 (AFB_1),to find out the responsible genes for hepatocellular carcinoma(HCC) and to further understand the underlying molecularmechanism.METHODS:Tree shrews (Tupaia belangerichinensis)weretreated with or without AFB_1 for about 90 weeks.Liverbiopsies were performed regularly during the animalexperiment.Eight shares of total RNA were respectivelyisolated from 2 HCC tissues,2 HCC-surrounding non-cancerous liver tissues,2 biopsied tissues at the early stage(30th week) of the experiment from the same animals asabove,1 mixed sample of three liver tissues biopsied at thebeginning (0th week) of the experiment,and another 1 mixedsample of two liver tissues from the untreated control animalsbiopsied at the 90th week of the experiment.The sampleswere then tested with the method of Atlas~(TM) cDNA microarrayassay.The levels of gene expression in these tissues takenat different time points during hepatocarcinogenesis werecompared.RESULTS:The profiles of differently expressed genes werequite different in different ways of comparison.At the sameperiod of hepatocarcinogenesis,the genes in the samefunction group usually had the same tendency for up-ordown-regulation.Among the checked 588 genes that wereknown to be related to human cancer,89 genes (15.1%)were recognized as“important genes”because they showedfrequent changes in different ways of comparison.Thedifferentially expressed genes during hepatocarcinogenesiscould be classified into four categories:genes up-regulatedin HCC tissue,genes with similar expressing levels in bothHCC and HCC-surrounding liver tissues which were higherthan that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue,and genes up-regulatedprior to the development of HCC but down-regulated afterthe development of HCC.CONCLUSION:A considerable number of genes couldchange their expressing levels both in HCC and in HCC-surrounding non-cancerous liver tissues.A few modulargenes were up-regulated only in HCC but not in surroundingliver tissues,while some apoptosis-related genes weredown-regulated in HCC and up-regulated in surroundingliver tissues.To compare gene-expressing levels amongthe liver tissues taken at different time points duringhepatocarcinogenesis may be helpful to locate theresponsible gene (s) and understand the mechanism forAFB_1 induced liver cancer.     

Expression of transforming growth factor-alpha and hepatitis B surface antigen in human hepatocellular carcinoma tissues and its significance

AIM:To evaluate the expression of transforming growthfactor-alpha (TGF-α) and hepatitis B surface antigen (HBsAg) in human hepatocellular carcinoma (HCC) tissues and its significance.METHODS:Seventy specimens of HCC tissues were detected by immunohistochemical method. Five specimens of normal human liver tissues were used as control.RESULTS: The TGF-o~ positive expression rates in HCC and its surrounding tissues were 74.3%(52/70) and 88.1%(52/59), respectively. TGF-α positive granules were mainly in the cytoplasm and fewer existed on the karyotheca. The TGF-α positive expressing rate in well differentiated HCC was significantly higher than that in moderately and poorly differentiated HCC (P&lt;0.05).The TGF-α positive expression also was observed in intrahepatic bile ducts (part of those were hyperplastic ducts).The HBsAg positive expression rates in HCC and its surrounding tissues were 21.4%(15/70) and 79.7%(47/59), respectively.HBsAg positive granules were in the cytoplasm, inclusion and on the karyotheca.There was a prominent positive correlation between TGF-α and HBsAg expression in HCC surrounding tissues (P&lt;0.05,γ=0.34). TGF-α was usually existed with HBsAg in regenerated and/or dysplastic liver cells.In the five normal liver tissues, TGF-α and HBsAg were not detectable in hepatocytes and bile ducts.CONCLUSION:Hepatitis B virus infection is dosely related with hepatocarcinogenesis.The overexpression of TGF-α in the liver seems to be associated with the regeneration of hepatocytes injured by HBsAg.The continued expression of TGF-α might lead to dysplasia of liver cells and development of HCC. Furthermore, TGF-α might play a role in morphogenesis and regeneration of intrahepatic bile ducts.     

Effect of hypoxia on expression of placental trophoblast cells SATB1 and β-catenin and its correlation with the pathogenesis of preeclampsia

Objective: To study the effect of hypoxia on the expression of placental trophoblast cells SATB1 and β-catenin and its correlation with the pathogenesis of preeclampsia. Methods: Trophoblastic cell lines HRT8/SVneo were cultured, SATB1 and β-catenin expression and cell biological behavior were determined after hypoxia reoxygenation treatment; cell biological behavior and the expression of related genes were determined after the transfection of SATB1 and β-catenin siR NA; preeclampsia placenta and normal placenta tissues were collected and the expression of SATB1 and β-catenin were determined. Results: OD value, cell migration rate, m RNA contents of SATB1 and β-catenin of H/R group were significantly lower than those of Nor group, cell apoptosis rate was higher than that of Nor group and the number of invasive cells was less than that of Nor group; OD value and bcl-2 mRNA content of SATB1-siRNA group were lower than those of NC group; cell apoptosis rate as well as Bax, Caspase-3, caspase-6 and caspase-9 mRNA contents were higher than those of NC group; cell migration rate as well as CTSB, CTSD, MMP2 and MMP9 mRNA contents of β-catenin-siRNA group were lower than those of NC group; the number of invasive cells was less than that of NC group; the expression levels of SATB1 and β-catenin in preeclampsia placenta tissue were significantly lower than those in normal placenta tissue. Conclusions: Hypoxia can inhibit the expression of SATB1 and β-catenin in the pathogenesis of preeclampsia, which can affect the proliferation, apoptosis, migration and invasion of cells.     

Expression of MUC1 and its significance in hepatocellular and cholangiocarcinoma tissue

AIM: To investigate the relation between MUC1 expression, distribution, and prognosis in hepatocellular and cholangiocarcinoma (HCC and CC) and cirrhotic liver tissues, and their significance in HCC and CC diagnosis. METHODS: Expression and distribution of MUC1 were examined by immunohistochemical assay with anti-MUC1 mAb in 59 samples of HCC and 37 samples of CC, 20 samples of cirrhotic liver tissues, and 10 samples of normal liver tissues, seeking possible associations between MUC1 positive expression, distribution in HCC and CC (primary liver cancer, PLC) cases and the studied clinical data. RESULTS: Immunohistochemical analysis of MUC1 expression showed that in the 96 PLC samples, 68 (70.8%) were strong positive, and 6 (6.2%) were weak positive. Only 4 in the 20 cirrhotic liver tissues were found to be weak positive, while no expression of MUC1 was detected in normal liver tissues. Apparently, the high expression rate of MUC1 in PLC tissues was statistically significant in comparison to that in cirrhotic and normal liver tissues. The expressed MUC1 protein, stained in dark brownish or brownish-yellow particles, chiefly localized on the cancer cell membranes or in cytoplasm. In the 68 strong positive samples, 40 were detected on cell membrane and the other 28 were in cytoplasm. In addition, follow-up studies of those PLC cases demonstrated that MUC1 expression on cell membrane or in cytoplasm was closely associated with PLC prognosis. The expression of MUC1 in PLC had little statistical significance in respect of the pathological types and sizes of the tumors, but a strong relationship regarding histological differentiation, metastasis of lymph nodes, portal canal emboli, and post-operational recurrence of the carcinomas. After 3 years of tumor excision, the metastasis rate in MUC1 positive expression group (67.6%) was much higher than that in MUC1 weak expression group (33.3%) and negative expression group (31.8%), and thus the survival rate in MUC1-positive expression group was significantly different from that in weak and negative expression groups. CONCLUSION: Expression and localization of MUC1 proteins in primary liver carcinomas (PLCs) may act as prognostic markers, and MUC1 molecules might be helpful in differential diagnosis.     

Preventive effect of Ganfujian granule on experimental hepatocarcinoma in rats

AIM:To investigate the inhibitory effect of dietary andmedicinal formula Ganfujian granule on diethylnitrosamine(DEN)-induced hepatocarcinoma in rats.METHODS:Male SD rats had free access to water containing0.1 g/L DEN for 16 weeks,during which the rats fed withstandard diet or administration of Ganfujian granule(30.4 g/Kgin diet).At weeks 4,8,12 and 16 of hepatocarcinogenesis5 rats of each group were sacrificed,and at week 20 another30 rats were sacrificed from each group.The end point forsurvival observation was at week 28.Immunochemistrymethods were used to examine the effect of Ganfujiangranule on the process of hepatocarcinogenesis includingproliferation of hepatocytes and cell cycle modulation.RESULTS:Ganfujian granule could reduce and delay theincidence of hepatocarcinoma in rats and prolong the survivalof animals.In addition,Ganfujian granule had a markedinhibitory effect on high expression of cyclin dependent kinase(CDK4)during the whole process of hepatocarcinogenesisand cyclin D1 at week 16 and the number of proliferatingcell nuclear antigen(PCNA)positive cells in different stagesof hepatocarcinogenesis.CONCLUSION:Ganfujian granule can reduce and delaythe incidence of hepatocarcinoma in rats by exerting director indirect effects on cell cycle and inhibiting uncontrolledproliferation of hepatocytes.     

Potential role of p53 mutation in chemical hepatocarcinogenesis of rats

AIM:Inactivation of p53 gene is one of the most frequentgenetic alterations in carcinogenesis.The mutation statusof p53 gene was analyzed,in order to understand theeffect of p53 mutation on chemical hepatocarcinogenesisof rats.METHODS:During hepatocarcinogenesis of rats inducedby 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB),prehepatocarcinoma and hepatocarcinoma loci werecollected by laser capture microdissection (LCM),andquantitatively analyzed for levels of p53 mRNA byLightCycler~(TM) real-time RT-PCR and for mutations in p53 geneexons 5-8 by direct sequencing.RESULTS:Samples consisting of 44 precancerous foci and24 cancerous foci were collected by LCM.A quantitativeanalysis of p53 mRNA showed that p53 mRNA peaked at anearly stage (week 6) in the prehepatocarcinoma lesion,morethan ten times that of adjacent normal tissue,and graduallydecreased from week 6 to week 24.The expression of p53mRNA in adjacent normal tissue was significantly lower thanthat in prehepatocarcinoma.Similar to prehepatocarcinoma,p53 mRNA in cancer was markedly higher than that inadjacent normal tissue at week 12,and was closer to normalat week 24.Direct p53 gene sequencing showed that 35.3%(24/68) (9 precancer,15 cancer) LCM samples exhibitedpoint mutations,20.5% of prehepatocarcinoma LCM samplespresented missense mutations at exon 6/7 or/and 8,andwas markedly lower than 62.5% of hepatocarcinoma ones(P<0.01).Mutation of p53 gene formed the mutant hot spotsat 5 codons.Positive immunostaining for p53 protein couldbe seen in prehepatocarcinoma and hepatocarcinoma fociat 24 weeks.CONCLUSION:p53 gene mutation is present in initialchemical hepatocarcinogenesis,and the mutation of p53gene induced by 3'-Me-DAB is an important factor ofhepatocarcinogenesis.