OBJECTIVETo explore the relationship between the alteration in GGT mRNA expression and the development of HCC.

METHODSThree GGT mRNA types (F, H, and P) in normal liver tissues, diseased liver tissues without HCC, cancerous and noncancerous tissues from livers with HCC, and noncancerous tissues from livers with metastatic tumor were tested by RT-PCR.

RESULTSIn normal livers, the main type of GGT mRNA was type F. In liver diseases but not HCC, the distribution of the type GGT mRNA was nearly the same as in normal livers. The prevalence of type H was significantly higher in both cancerous and noncancerous tissues of livers with HCC than in livers without HCC (P<0.05). The prevalence of type F in cancerous tissues was significantly lower than that in livers without HCC (P<0.05).

CONCLUSIONSThe GGT mRNA expression in the human liver will shift from type F to type H during the development of HCC. The fragment analysis of GGT genes may be a sensitive assay to detect hepatic cell canceration.

Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Female ; Gene Expression Regulation, Enzymologic ; Humans ; Liver ; enzymology ; metabolism ; pathology ; Liver Diseases ; enzymology ; genetics ; pathology ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Male ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; gamma-Glutamyltransferase ; genetics

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; enzymology ; pathology ; virology ; Hepacivirus ; genetics ; Humans ; Liver Neoplasms ; enzymology ; pathology ; virology ; Telomerase ; metabolism ; Viral Core Proteins ; genetics ; metabolism

OBJECTIVETo investigate the expression of heparanase mRNA and its relation with the clinicopathological features and angiogenesis in primary hepatocellular carcinoma (HCC).

METHODSExpression of heparanase mRNA was detected by RT-PCR in 51 HCC lesions, and microvessel density (MVD) was detected by immunohistochemical stain with a factor VIII-related monoclonal antibody.

RESULTSExpression of heparanase mRNA was shown in 49.0% (25/51) HCC lesions. The positive rate of heparanase expression in tumors larger than 3 cm (63.6%, 21/33) was significantly higher than those in smaller tumors (22.2%, 4/18; P < 0.01). Heparanase expression was more frequent in highly invasive tumors (70.0%, 14/20) compared with moderately invasive tumors (46.7%, 7/15) and low invasive ones (25.0%, 4/16; P < 0.05). Moreover, heparanase expression in tumors with high MVD (62.5%, 20/32) was significantly higher than those in tumors with low MVD (26.3%, 5/19; P < 0.05).

CONCLUSIONHeparanase mRNA expression may be important for the growth, invasion and angiogenesis of hepatocellular carcinoma.

Adult ; Carcinoma, Hepatocellular ; blood supply ; enzymology ; pathology ; Female ; Glucuronidase ; biosynthesis ; genetics ; Humans ; Liver ; enzymology ; Liver Neoplasms ; blood supply ; enzymology ; pathology ; Male ; Microcirculation ; pathology ; Middle Aged ; Neoplasm Invasiveness ; Neovascularization, Pathologic ; enzymology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Burden

Apoptosis ; physiology ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Carrier Proteins ; biosynthesis ; genetics ; DNA-Binding Proteins ; metabolism ; Gene Targeting ; Humans ; Liver Neoplasms ; enzymology ; pathology ; RNA Interference ; Telomerase ; metabolism ; Tumor Cells, Cultured

NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition, NF-kappaB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.
Animals ; Carcinoma, Hepatocellular/*enzymology/genetics/pathology ; Cell Line ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Humans ; Liver/*enzymology/metabolism/pathology ; Liver Neoplasms/*enzymology/genetics/pathology ; Mice ; Mice, Nude ; NIH 3T3 Cells ; NM23 Nucleoside Diphosphate Kinases/*genetics/metabolism

OBJECTIVETo construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatocellular carcinoma cells.

METHODSThe sequence of the siRNA targeting hTERT, U6 promoter and EGFP gene were amplified by PCR and inserted into the mammalian retroviral expression vector pLXSN to construct the recombinant retroviral vector pLXSN-EGFP-U6-siTERT. The vector was then used to infect human hepatocellular carcinoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by MTT assay.

RESULTSSequence analysis and restriction enzyme digestion showed confirmed successful construction of the recombinant expression vector pLXSN-EGFP-U6-siTERT. The telomerase activity of the infected HepG2 cells was reduced by 23.84%, 58.03% and 85.01% at 24, 48 and 72 h after the infection, respectively (P<0.05). The cell apoptosis rate of the infected cells was 29.05% at 24 h after the infection. The cell proliferation was markedly inhibited by the infection with the vector in comparison to that of the control group.

CONCLUSIONhTERT siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells.

Apoptosis ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms ; enzymology ; genetics ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Retroviridae ; genetics ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVETo study focal adhesion kinase (FAK) expression in hypoxia-stressed SMMC-7721 cells and the role of FAK expression in the hypoxia-induced invasion of SMMC-7721 cells.

METHODSSMMC-7721 cells were cultured in 21% O2 or 1% O2. FAK expression was determined by Western blot. The siRNA expression vector pshRNA-FAK targeting to FAK and the control vector pGensil-2 were transfected into SMMC-7721 cells. The hypoxia-induced migration and invasion ability of SMMC-7721 cells transfected with pshRNA-FAK were analyzed. In normoxia, invasion of SMMC-7721 cells transfected with pcDNA3-FAK was analyzed.

RESULTSThe expression of FAK was increased significantly in SMMC-7721 cells 24 h after hypoxia stress (P<0.01). The level of FAK protein was decreased by 74.6%+/-5.1% after the pshRNA-FAK transfection in normoxia and hypoxia. The migration and invasion of SMMC-7721 cells was increased in 1% O2 (P<0.01). However, the migration and invasion of SMMC-7721 cells transfected with pshRNA-FAK was decreased in 1% O2 (P<0.05). Overexpression of FAK significantly stimulated the invasion of SMMC-7721 cells.

CONCLUSIONUp-regulation of FAK may play an important role in the invasion of SMMC-7721 cells induced by hypoxia.

Blotting, Western ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Humans ; Hypoxia ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Neoplasm Invasiveness ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the expression and significance of nitric oxide synthase (cNOS) mRNA in primary hepatocellular carcinoma (HCC), cirrhotic liver and normal liver tissue.

METHODScNOS mRNA expression in 80 HCC, 40 cirrhotic liver and 20 normal liver tissue were observed by in situ hybridization. CD34 immunostain was used to measure the microvascular density (MVD) and Ki-67 immunostain to proliferative index.

RESULTSExpression of cNOS mRNA was observed in the liver cancer cells, endothelial cells in the non-cancerous liver tissues and mononuclear and/or phagocytes. Expression of cNOS mRNA in tumor cells of HCC was higher than that in the liver cells of cirrhotic liver (P < 0.01) which was higher than the normal liver tissue. Expression in the endothelial cells was higher in HCC and cirrhotic liver than those in the normal liver tissue (P < 0.01). HCC with positive cNOS mRNA expression in the endothelial cells showed higher extent of neovascularization and degree of proliferative index. The more MVD, the higher proliferative index, which increased in metastatic tumors.

CONCLUSIONcNOS mRNA expression was involved in oncogenesis, angiogenesis and progression of hepatocellular carcinoma.

Adolescent ; Adult ; Aged ; Carcinoma, Hepatocellular ; blood supply ; enzymology ; pathology ; Cell Proliferation ; Child ; Female ; Humans ; Liver ; blood supply ; enzymology ; Liver Neoplasms ; blood supply ; enzymology ; pathology ; Male ; Middle Aged ; Neovascularization, Pathologic ; etiology ; Nitric Oxide Synthase ; genetics ; physiology

OBJECTIVESTo investigate the apoptosis induced by antisense oligodeoxynucleotide (ASODN) against survivin and the mechanisms after the hepatocellular carcinoma SMMC-7721 cells transfected with the ASODN.

METHODSThe ASODN was transfected into SMMC-7721 cells mediated by liposomal reagent. The changes of cell cycle and apoptotic rate were detected by flow cytometry. The changes of cell skeleton was observed through confocal microscope. The activity of p38MAPK and caspase-3 were detected by immuno-precipitation and kinase activity assess methods, respectively.

RESULTSThere were control, sense control, 400, 600, 800, and 1 000 ng/ml ASODN groups (I - VI). The apoptotic rats were 0.70%, 0.76%, 2.43%, 7.82%, 23.11%, and 31.35% in groups I - VI, respectively, which in the ASODN-transfected groups were higher than that in the control group (tor=20.9, P<0.01). The activity of p38MAPK increased significantly, when the ASODN was transfected at the concentration of 600 ng/ml or more, so did the caspase-3 activity (the p38MAPK and caspase-3 activity in groups I - VI were 7.03, 7.07, 13.47, 16.37, 43.97, 47.87 and 0.015+/-0.010, 0.014+/-0.002, 0.026+/-0.003, 0.042+/-0.001, 0.093+/-0.001, 0.100+/-0.001, respectively).

CONCLUSIONSASODN targeting at survivin mRNA can induce G2/M stop, activate p38MAPK and caspase-3. The activated caspase-3 destroys the cell skeleton microfilament system, resulting in apoptosis.

Apoptosis ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Microtubule-Associated Proteins ; antagonists & inhibitors ; genetics ; Neoplasm Proteins ; Oligonucleotides, Antisense ; therapeutic use

To examine the differential expression pattern of Ech1 protein in mouse Hca-F and Hca-P hepatocarcinoma cell lines with high and low rates of lymphatic metastasis, respectively, and to investigate the relationships between Ech1 expression and adhesion of Hca-F cells. Fluorescence two-dimensional difference in-gel electrophoresis (2D DIGE) and mass spectrometry were used to detect Ech1 expression. Ech1 gene silencing was achieved by stable transfection of Hca-F cells with a plasmid vector harboring short hairpin RNA (shRNA) targeting Ech1, pGPU6/GFP/Neo-shRNA-Ech1. Ech1 mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Adhesive properties of cells were assessed by hematoxylin-eosin staining and fluorimetric detection of extracellular matrix (ECM) proteins. Endogenous Ech1 protein level was remarkably higher in the highly metastatic Hca-F cell line than in the Hca-P cell line (2.7-fold by 2D DIGE; 1.5-fold by Western blotting). shRNA-induced silencing of Ech1 significantly reduced the adhesion ability of Hca-F cells, as evidenced by decreased absorbance values of fibronectin and collagen I (Hca-F cells vs. pGPU6/GFP/Neo-shRNA-Ech1 cells: 1.42+/-0.26 vs. 1.01+/-0.27 and 1.14+/-0.07 vs. 0.90+/-0.09, respectively; P less than 0.05). Down-regulation of Ech1 can inhibit the adhesive capacity of metastatic Hca-F cells.
Animals ; Carbon-Carbon Double Bond Isomerases ; genetics ; metabolism ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Down-Regulation ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms ; enzymology ; genetics ; pathology ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Mice ; Plasmids ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; pharmacology ; Transfection