Detection of PRL-2 gene expression in hepatocellular carcinoma by real-time fluorescence quantitative PCR

Objective:To quantitatively detect the expression level of PRL-2 in primary hepatocellular carcinoma using real-time fluorescence quantitative PCR.Methods:Total RNA isolated from human HCC and liver tissue adjacent to the tumor was reversely transcribed into cDNA.Real-time fluorescence quantitative PCR(Q-PCR) method was used to analyze the expres-sion level of PRL-2 gene.Results:The Q-PCR method was performed successfully to precisely detect RNA level.PRL-2 was expressed in all portal vein tumor thrombosis(PVTT) and HCC,but only in some paratumor tissue.The highest expression level of PRL-2 gene was recorded in PVTT;meanwhile expression level of PRL-2 was higher than that in paratumor liver tis-sues and in HCC(P < 0.01),and it was higher in HCC than that in paratumor liver tissues.Conclusion:The Q-PCR may be the most precise method to quantitatively detect RNA level and can be used in gene expression changes.The PRL-2 gene has higher expression in PVTT than that in HCC and in paratumor liver tissue cells,indicating that it plays an important role in the development and metastasis of the HCC.

Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21^(WAF1) expression and hepatic apoptosis

AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21WAF1gene expression and HCC apoptosis.METHODS In situ hybridization was employedto determine caspase 3 and p21WAF1expression inHCC.In situ end-labeling was used to detecthepatocytic apoptosis in HCC.RESULTS Twenty-one of 39（53.8%）cases ofHCC were found to express caspase 3transcripts,while 45.2% of HCC failed toexpress caspase 3.Non-cancerous adjacent livertissues showed more positive caspase 3（87.5%,7/8）as compared with HCC（P【0.05）.The expression of caspase 3 is correlated withHCC differentiation,72.2%（13/18）ofmoderately to highly differentiated HCC showedcaspase 3 transcripts positive,while only 38.1%of poorly differentiated HCC harbored caspase 3transcripts（P【0.05）.No relationship wasfound between caspase 3 expression and tumorsize or grade or metastasis,although 52.5%（5/8）of HCC with metastasis were caspase 3positive and a little higher than that with nometastasis（51.6%,P】0.05）.Expression of caspase 3 alone did not affect the apoptosisindex（AI）of HCC.The AI was 7.12%o in caspase3-positive tumors（n=21）,while in caspase 3-negative cases（n=18）6.59%0（P】0.05）.Expression of caspase 3 clearly segregated withp21WAF1positive tumors as compared withp21WAF1-negative cases（16 of 23,69.6% versus5 of 16,31.3%）with statistical significance（P=0.017）.In the cases with positive caspase 3and negative p21WAF1,the Al was found slightlyhigher,but with no statistical significance,thanthat with expression of p21WAF1and caspase 3（7.21‰ vs 6.98‰,P】0.05）.CONCLUSION Loss of caspase 3 expressionmay contribute to HCC carcinogenesis,althoughthe expression of caspase 3 does not correlatewell with cell apoptosis in HCC.p21WAF1may bemerely one of the inhibitors which can reducecaspase 3 mediated cell apoptosis in HCCs.

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

Monitoring microarray-based gene expression profile changes in hepatocellular carcinoma

AIM: To find out key genes responsible for hepatocarc inogenesis and to further understand the underlying molecular mechanism through investigating the differential gene expression between human normal liver tissue and hepatocellular carcinoma (HCC).METHODS: DNA microarray was prepared by spotting PCR products of 1 000 human genes including 445 novel genes, 540 known genes as well as 12 positive (housekeeping) and 3 negative controls (plant gene) onto treated glass slides. cDNA probes were prepared by labeling normal liver tissue mRNA and cancer liver tissue mRNA with Cy3-dUTP and Cy5-dUTP separately through reverse transcription. The arrays were hybridized against the cDNA probe and the fluorescent signals were scanned. The dataobtained from repeated experiments were analyzed. RESULTS: Among the 20 couple samples investigated (from cancerous liver tissue and normal liver tissue), 38 genes including 21 novel genes and 17 known genes exhibited different expressions. CONCLUSION: cDNA microarray technique is powerful to identify candidate target genes that may play important roles in human carcinogenesis. Further analysis of the obtained genes is helpful to understand the molecular changes in HCC progression and ultimately may lead to the identification of new targets for HCC diagnosis and intervention.

Difference in hTERT Gene Expressions between HbsAg-Positive and HbsAg-Negative Hepatocellular Carcinoma

To investigate the difference in expression of hTERT gene between HbsAg-positive human hepatocellular carcinoma (HCC) and HbsAg-negative HCC and to explore the relationship between HBV infection and hTERT gene expression in HCC. The expression of hTERT protein in 30 cases of HbsAg positive HCC and 17 cases of HbsAg negative HCC was detected by immunohistochemistry (SP method), and the expression of hTERT mRNA was analyzed by reverse transcription polymerase chain reaction (RT-PCR). t-test, Chi-squared test and cochran- armitage trend test were used to see whether there was an interrelation between HBsAg and hTERT gene in HCC. The expression of hTERT protein was mostly located in plasm and occasionally in the nucleus of liver cancer cells. The positive rate of hTERT protein and hTERT mRNA in HbsAg positive HCC- 93.33 % (28/30) and 83.33 % (25/30) respectively which were much higher than those in HbsAg negative HCC- 52.94 % (9/17), 47.06 % (8/17) (P<0.01) respectively. HbsAg is related to hTERT gene expression in human hepatocellular carcinoma. The hTERT gene activated by the efficacious ingredient of HBV may play an important role in hepatocellular transformation and carcinogenesis.

KAI1/CD82 gene expression in benign prostatic hyperplasia and late-stage prostate cancer in Chinese

Aim: To evaluate KAII/CD82 expression in Chinese patients with benign prostatic hyperplasia (BPH) and late-stage carcinoma of prostate (CaP). Methods: Thirty Chinese patients with benign prostatic hyperplasia and 34 withCaP (adenocarcinoma clinical stage C and D) were analyzed by means of immunohistochemical methods. Results:The KAII/CD82 expression in BPH tissue was all positive, which was uniformly located on the glandular cell mem-brane at the cell-to-cell borders, but KAII/CD82 expression in metastasis CaP tissues was either significantly lower thanthat of BPH or negative, and the immunostaining pattern was not continuous. In late-stage CAP KAII/CD82 expressionwas correlated inversely to the pathological grade ( P < 0.05), but not to clinical stage ( P > 0.05). Conclusion:The authors believe that decreased and negative KAII/CD82 expression in late-stage CaP may be related to tumor pro-gression and metastasis, and appears to be a prognostic marker.

Microsatellite instability,MMR gene expression and proliferation kinetics in colorectal cancer with famillial predisposition

INTRODUCTIONGenetic instability is a conunon property of manyhuman cancers,including those of HNPCC.A novel form of genetic instability involving somaticalterations,such as deletions and insertions insimple repeated sequences,has been found.Microsatellitcs are relatively short runs of tandemlyrepeated sequences scattered throughout

Challenges of incorporating gene expression data to predict HCC prognosis in the age of systems biology

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide.The recurrence of HCC after curative treatments is currently a major hurdle.Identification of subsets of patients with distinct prognosis provides an opportunity to tailor therapeutic approaches as well as to select the patients with specific sub-phenotypes for targeted therapy.Thus,the development of gene expression profiles to improve the prediction of HCC prognosis is important for HCC management.Although several gene signatures have been evaluated for the prediction of HCC prognosis,there is no consensus on the predictive power of these signatures.Using systematic approaches to evaluate these signatures and combine them with clinicopathologic information may provide more accurate prediction of HCC prognosis.Recently,Villanueva et al developed a composite prognostic model incorporating gene expression patterns in both tumor and adjacent tissues to predict HCC recurrence.In this commentary,we summarize the current progress in using gene signatures to predict HCC prognosis,and discuss the importance,existing issues and future research directions in this field.

NORTHERN BLOT ANALYSIS OF nm23 GENE EXPRESSION IN HUMAN LUNG CANCER

Objective: To investigate the role of nm23 gene expression in human lung cancer. Methods: Forty human lung cancer tissues and 19 non-cancer pulmonary tissues were studied for their nm23-H1 and nm23-H2 mRNA expression with non-radioactive Northern blot hybridization. The correlation of nm23 mRNA expression with clinical features of lung cancer was analyzed. Results: The mRNA expression of nm23-H2 gene in poorly differentiated squamous cell carcinoma was significantly decreased compared to that in moderate-high differentiated squamousd cell carcinoma. The mRNA expression of nm23-H1 and nm23-H2 gene in small cell lung cancer was significantly decreased compared to that in squamous cell carcinoma. No significant difference in nm23 mRNA expression was observed between lung cancer with and without lymph node metastasis, nor was there significant difference between tumor stage. Conclusion: The mRNA expression of nm23 gene is correlated with the degree of differentiation of lung cancer, but there is no evidence of metastasis suppression effect by nm23 gene.

Hepatocellular carcinoma:An analysis of the expression status of stress granules and their prognostic value

BACKGROUND Hepatocellular carcinoma(HCC)is a global popular malignant tumor,which is difficult to cure,and the current treatment is limited.AIM To analyze the impacts of stress granule(SG)genes on overall survival(OS),survival time,and prognosis in HCC.METHODS The combined The Cancer Genome Atlas-Liver Hepatocellular Carcinoma(TCGA-LIHC),GSE25097,and GSE36376 datasets were utilized to obtain genetic and clinical information.Optimal hub gene numbers and corresponding coefficients were determined using the Least absolute shrinkage and selection operator model approach,and genes for constructing risk scores and corresponding correlation coefficients were calculated according to multivariate Cox regression,respectively.The prognostic model’s receiver operating characteristic(ROC)curve was produced and plotted utilizing the time ROC software package.Nomogram models were constructed to predict the outcomes at 1,3,and 5-year OS prognostications with good prediction accuracy.RESULTS We identified seven SG genes(DDX1,DKC1,BICC1,HNRNPUL1,CNOT6,DYRK3,CCDC124)having a prognostic significance and developed a risk score model.The findings of Kaplan-Meier analysis indicated that the group with a high risk exhibited significantly reduced OS in comparison with those of the low-risk group(P<0.001).The nomogram model’s findings indicate a significant enhancement in the accuracy of OS prediction for individuals with HCC in the TCGA-HCC cohort.Gene Ontology and Gene Set Enrichment Analysis suggested that these SGs might be involved in the cell cycle,RNA editing,and other biological processes.CONCLUSION Based on the impact of SG genes on HCC prognosis,in the future,it will be used as a biomarker as well as a unique therapeutic target for the identification and treatment of HCC.

Identification of key genes underlying clinical features of hepatocellular carcinoma based on weighted gene co‑expression network analysis and bioinformatics analysis

Objective: To identify module genes that are closely related to clinical features of hepatocellular carcinoma (HCC) by weighted gene co‑expression network analysis, and to provide a reference for early clinical diagnosis and treatment. Methods: GSE84598 chip data were downloaded from the GEO database, and module genes closely related to the clinical features of HCC were extracted by comprehensive weighted gene co‑expression network analysis. Hub genes were identified through protein interaction network analysis by the maximum clique centrality (MCC) algorithm;Finally, the expression of hub genes was validated by TCGA database and the Kaplan Meier plotter online database was used to evaluate the prognostic relationship between hub genes and HCC patients. Results: By comparing the gene expression data between HCC tissue samples and normal liver tissue samples, a total of 6 262 differentially expressed genes were obtained, of which 2 207 were upregulated and 4 055 were downregulated. Weighted gene co‑expression network analysis was applied to identify 120 genes of key modules. By intersecting with the differentially expressed genes, 115 candidate hub genes were obtained. The results of enrichment analysis showed that the candidate hub genes were closely related to cell mitosis, p53 signaling pathway and so on. Further application of the MCC algorithm to the protein interaction network of 115 candidate hub genes identified five hub genes, namely NUF2, RRM2, UBE2C, CDC20 and MAD2L1. Validation of hub genes by TCGA database revealed that all five hub genes were significantly upregulated in HCC tissues compared to normal liver tissues;Moreover, survival analysis revealed that high expression of hub genes was closely associated with poor prognosis in HCC patients. Conclusions: This study identifies five hub genes by combining multiple databases, which may provide directions for the clinical diagnosis and treatment of HCC.

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.

Construction of retroviral vectors to induce a strong expression of human class Ⅰ interferon gene in human hepatocellular carcinoma cells in vitro

AIM To establish the hepatoma cell specific expression of human interferon gene mediated by retroviral vectors. METHODS Human interferon α and interforon β complementary DNA (IFNs cDNA) were cloned into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNA and pMNSIFNB, where the transcription of IFN gene was driven by SV40 early region promoter, and pMNAIFNA, pAMNSIFNA and pMNAIFNB, where the transcription of IFN gene was driven by SV40 early region promoter regulated by α fetoprotein enhancer. The retroviral constructs were respectively introduced into retroviral amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection rate was (4～40)×10 3 colonies/μg DNA/10 6 PA317 cells. The retrovirus infection rate was (5～500)×10 4 colony forming units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells and melanoma cell lines in the presence of 4mg/L polybrene. RESULTS Northern and Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α fetoprotein enhancer induced efficient and apecific transcription and expression of IFNs gene driven by the promoter of different origin in human hepatoma cells by which α fetoprotein was highly produced. CONCLUSION Cis active element of α fetoprotein gene can drive specific expression of IFNs gene in human hepatoma cells, which provides some valuable data for the hepatoma specific immune gene therapy.

Expression of gap junction genes connexin 32,connexin 43 and their proteins in hepatocellular carcinoma and normal liver tissues

AIM To investigate the significance andmechanism of cx32 mRNA,cx43 mRNA and theirproteins in hepatocarcinogenesis.METHODS Sixty-one cases of HCC and 14cases of normal liver tissues were detected byimmunohistochemical and in situ hybridization(ISH)methods.RESULTS In HCC grades Ⅰ,Ⅱ,Ⅲ and normalliver tissues,the positive rates of Cx32 proteinwere 55.6%,42.1%,18.2% and 92.9%,respectively.The detection rates of Cx43 proteinwere 44.4%,26.3%,12.1% and 78.6%,respectively.There was significant difference inCx32 and Cx43 protein between HCC and normalliver tissues(P【0.01).ISH the positive rates ofcx32 mRNA shown by ISH in HCC grades Ⅰ,Ⅱ,Ⅲ and normal liver tissues were 88.9%,84.2%,87.9% and 92.9%,respectively.Those of cx43mRNA were 77.8%,78.6%,78.8% and 85.7%,respectively.There was no statistical differencein the positive rates of cx32 mRNA and cx43mRNA between HCC and normal liver tissue(P】0.05).CONCLUSION The aberrant location of Cx32and Cx43 proteins could be responsible forprogression of hepatocarcinogenesis,and thedefect of cx genes in post-translationalprocessing might be the possible mechanism.

Expression of nm23 gene in hepatocellular carcinoma tissue and its relation with metastasis

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Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

Gene Therapy of HSV-TK Transferred by the EBV based Expression Vector on Experimental Hepatocellular Carcinoma

To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.

EXPRESSION OF CELLULAR ONCOGENES IN HUMAN PRIMARY HEPATOCELLULAR CARCINOMA

With DNA-mRNA hybridization in situ technique, the expression of five oncogenes, c-N-ras, c-Ki-ras. c-Ha-ras, c-myc and c-fos, was observed in two cases of human hepatocellular carcinoma. The expression of c-N-ras & c-fos was greatly enhanced in tumor tissues of the two cases, and about 25% -50% of the tumor cells showed positive expression. The other three oncogenes namely c-Ki-ras, c-Ha-ras & c-myc, were not detected in these two carcinomas or in the non-cancerous liver tissues adjacent to the carcinomas. It is surmised that c-N-ras and c-fos may play coordinative role in maintaining the malignant phenotype of human primary hepatocellular carcinoma.

THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.