RNA, Messenger ; biosynthesis ; genetics ; RNA, Viral ; chemistry ; genetics ; Ribonucleases ; biosynthesis ; genetics ; Virion ; chemistry ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; Eukaryotic Cells/metabolism ; Genetic Vectors ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; alpha-Fetoproteins/*biosynthesis ; alpha-Fetoproteins/genetics

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths and the fifth most common cancer worldwide. Clinically therapeutic options for HCC are very limited, and the overall survival rate of patients is very low. Therefore, early diagnosis and treatment of HCC have important impact on overall survival of patients. At present, alpha-fetoprotein (AFP) is one of the most widely used serological markers for HCC. Many evidences have shown that as a specific onco-protein, AFP has great research value in the occurrence, development, diagnosis and treatment of HCC. Here, we briefly introduce the molecular mechanism of AFP in the regulation of HCC occurrence and development, and its role in tumor escape from immune surveillance. We focus on the application of AFP as an important HCC target or carcino-embryonic antigen (CEA) in HCC clinical diagnosis and treatment.
Biomarkers, Tumor/genetics* ; Carcinoma, Hepatocellular/therapy* ; Early Detection of Cancer ; Humans ; Liver Neoplasms/therapy* ; alpha-Fetoproteins

Objective: To explore carnosine dipeptidase 1 (CNDP1) potential value as a diagnostic and prognostic evaluator of hepatocellular carcinoma (HCC). Methods: A gene chip and GO analysis were used to screen the candidate marker molecule CNDP1 for HCC diagnosis. 125 cases of HCC cancer tissues, 85 cases of paracancerous tissues, 125 cases of liver cirrhosis tissues, 32 cases of relatively normal liver tissue at the extreme end of hepatic hemangioma, 66 cases from serum samples of HCC, and 82 cases of non-HCC were collected. Real-time fluorescent quantitative PCR, immunohistochemistry, western blot, and enzyme-linked immunosorbent assay were used to detect the differences in mRNA and protein expression levels of CNDP1 in HCC tissue and serum. Receiver operating characteristic (ROC) curves and Kaplan-Meier survival were used to analyze and evaluate the value of CNDP1 in the diagnosis and prognosis of HCC patients. Results: The expression level of CNDP1 was significantly reduced in HCC cancer tissues. The levels of CNDP1 were significantly lower in the cancer tissues and serum of HCC patients than those in liver cirrhosis patients and normal controls. ROC curve analysis showed that the area under the curve of serum CNDP1 in the diagnosis of HCC patients was 0.753 2 (95% CI 0.676-0.830 5), and the sensitivity and specificity were 78.79% and 62.5%, respectively. The combined detection of serum CNDP1 and serum alpha-fetoprotein (AFP) significantly improved the diagnostic accuracy (AUC = 0.820 6, 95% CI 0.753 5-0.887 8). The diagnostic sensitivity and specificity of serum CNDP1 for AFP-negative HCC patients were 73.68% and 68.75% (AUC = 0.793 1, 95% CI 0.708 8-0.877 4), respectively. In addition, the level of serum CNDP1 distinguished small liver cancer (tumor diameter < 3 cm) (AUC = 0.757 1, 95% CI 0.637 4-0.876 8). Kaplan-Meier survival analysis showed that CNDP1 was associated with a poor prognosis in HCC patients. Conclusion: CNDP1 may be a potential biomarker for the diagnostic and prognostic evaluation of HCC, and it has certain complementarity with serum AFP.
Humans ; Carcinoma, Hepatocellular/genetics* ; Liver Neoplasms/pathology* ; Prognosis ; Carnosine ; alpha-Fetoproteins/metabolism* ; Biomarkers, Tumor/genetics* ; Liver Cirrhosis/diagnosis* ; ROC Curve

OBJECTIVETo explore hepatitis C virus (HCV) non-structural protein 5A (NS5A)'s influence on inhibition of AFP expression executed by p53 protein and its possible molecular mechanism.

METHODSPlasmid transfection and MEIA were employed to observe p53's inhibitive effect on AFP expression of Huh7 cells and the HCV NS5A's influence on p53 function. Western blot was employed to find out if HCV NS5A affects p53 protein expression and GST pull down assay was applied to examine the interaction between HCV NS5A and p53.

RESULTSThe AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14322+/-2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13843+/-3218) ng/ml; no significant difference existed between these two groups (t = 1.42, P > 0.05). After transfection with pC53-NS3, the AFP level was decreased to (10 241+/-1326) ng/ml, and in comparison to the above two groups it had a statistically significant difference (t values were 2.41 and 2.38, P < 0.05). When co-transfected with pCNS5A and pC53-NS3, the AFP expression (14582+/-1238) ng/ml returned to the level of pRc/CMV transfected, and there was a remarkably significant difference between this and that of the pC53-NS3 transfected cells (t = 3.12, P < 0.01). HCV NS5A had no function on the p53 protein expression with Western blot experiment. In the GST pull down assay, an HCV NS5A protein band was found after GST-p53 was added, but not detected with GST only.

CONCLUSIONWe found that p53 has an inhibitive function on the AFP expression in Huh7 cells and HCV NS5A minimized this p53 function. HCV NS5A did not affect p53 protein expression, but was able to form a complex with p53, by which HCV NS5A inactivated this p53 function.

Carcinoma, Hepatocellular ; metabolism ; virology ; Hepacivirus ; genetics ; Humans ; Liver Neoplasms ; metabolism ; virology ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; genetics ; pharmacology ; Viral Nonstructural Proteins ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

To comparatively investigate ultrastructural characteristics and expressions of AFP (alpha-fetoprotein) and Tn (Thomsen-Friedenreich-related antigen) protein in AFP negative (AFP-) and AFP positive (AFP+) primary hepatocellular carcinoma. Fourty-three cases of AFP- and AFP+ hepatocellular carcinoma (HCC) tissues and five cases of normal liver tissues were divided into three groups: control group (normal liver tissue, n=5); AFP+ HCC group (the serum AFP level was higher than 10 ng/ml, n = 22); AFP- HCC group (the serum AFP level was lower than 10 ng/ml, n=21). The ultrastructural morphology was studied by transmission electron microscopy, the expressions of AFP and Tn protein were detected by immunohistochemistry and cell image analysis. 1. The immunohistochemical study showed that (1) the expression intensity and positive rate of Tn protein in AFP- HCC group were markedly higher than that in AFP+ HCC group (P<0.01); (2) The expression intensity of AFP in AFP- HCC group was lower than that in AFP+ HCC group (P<0.01). 2. The transmission electron microscopy demonstrated that some AFP- HCC cells linked closely with each other, others dispersed loosely just as cultured cells, the remarkable morphologic features in AFP- HCC cells were simple organelles, but they were abundant in the free polyribosomes. In AFP+ HCC group, all the HCC cells linked closely together and were rich organelles in their cytoplasm, especially the rough endoplasmic reticula. In addition, mitochondria and Golgi complex were obviously observed. (1) The AFP and Tn protein had discrepancy distribution in AFP- and AFP+ HCC tissues, Tn protein may be one of the early diagnostic indicators in AFP- HCC; (2) The synthetic locations of the AFP and Tn protein were different in hepatocarcinoma cells by ultrastructural observation.
Antigens, Tumor-Associated, Carbohydrate ; biosynthesis ; genetics ; Biomarkers, Tumor ; Carcinoma, Hepatocellular ; metabolism ; ultrastructure ; Female ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; ultrastructure ; Male ; Tumor Cells, Cultured ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo establish a new extrahepatic growing hepatocellular carcinoma cell line.

METHODSA specimen from extrahepatic growing hepatocellular carcinoma was cultured in vitro. Cancer cells were studied morphologically and subjected to karyotype analysis, DNA analysis, and tumor formation evaluation.

RESULTSMorphological observation and functional analysis showed that their features were similar to those of HCC. Chromosomes with a variation of 76 approximately 104 were able to secret AFP in vitro and to form bile canaliculi with microvilli.

CONCLUSIONEGHC-9901 cell line has characteristics of the extrahepatic growing hepatocellular carcinoma.

Adult ; Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; Chromosome Aberrations ; Humans ; Liver Neoplasms ; genetics ; pathology ; Male ; Mice ; Mice, Nude ; Tumor Cells, Cultured ; alpha-Fetoproteins ; analysis

OBJECTIVETo detect and compare the transcriptional activities of tumor-specific survivin and AFP promoters in various hepatocellular carcinoma cell lines and to lay some groundwork for targeting gene therapy in human hepatocellular carcinoma.

METHODSThe fragment of survivin and AFP promoters were acquired by PCR amplification and were cloned into the reporter plasmid pGL3-Basic, which contained a luciferase gene. The constructed eukaryotic expression plasmid pGL3-SUR and pGL3-AFP, in which the expression of the luciferase was derived by survivin or the AFP promoter, were transfected into three HCC cell lines. At 24 hours post transfection (p.t.), the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System. A pGL3-CMV, containing the CMV promoter controlled luciferase gene, was used as a positive control.

RESULTSBoth survivin and AFP promoters had transcriptional activities in all three HCC cell lines and the transcriptional activity of the survivin promoter was much higher than the AFP promoter (52-98 times) and reached a level of 16% approximately 21% of the transcriptional activity of the CMV promoter.

CONCLUSIONOur data reveals that the survivin promoter possesses a high transcriptional activity in all three established HCC cell lines and may serve as a useful tool for transcriptional targeting gene therapy of HCCs.

Carcinoma, Hepatocellular ; genetics ; Gene Targeting ; Genetic Therapy ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; genetics ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Promoter Regions, Genetic ; genetics ; Transcription, Genetic ; Transfection ; Tumor Cells, Cultured ; alpha-Fetoproteins ; genetics

To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
Animals ; CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo investigate the relationship between serum alpha-fetoprotein (AFP) variant levels in patients with hepatocellular carcinoma (HCC) and cancer cells disseminating through blood.

METHODSSerum AFP variant levels were measured by crossed immunoaffino-electrophoresis in the presence of lectin before initial surgical treatment in HCC patients. Circulating tumor cells were simultaneously detected in pre-operative blood samples using reverse transcription-polymerase chain reaction (RT-PCR) for AFP mRNA.

RESULTSForty-six HCC patients with serum AFP positive were studied. Serum AFP variant level > or 20% was showed in 37 patients, among whom there were 22 (59.5%) showing AFP mRNA positive. In contrast, the positive AFP mRNA expression was only observed in 2 out of 9 patients (22.2%) with AFP variant level<20% (x(2)=4.02, P<0.05).

CONCLUSIONIn hepatocellular carcinoma patients, increased AFP variant levels are associated with a haematogenous spread of tumor cells.

Adult ; Aged ; Carcinoma, Hepatocellular ; blood ; Female ; Humans ; Liver Neoplasms ; blood ; Male ; Middle Aged ; RNA, Messenger ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; alpha-Fetoproteins ; analysis ; genetics