Re-expression of RASSF1A by 5-Aza-CdR Induced Demethylation of the Promoter Region in Human Biliary Tract Carcinoma Cells

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2’-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 μmol/L for 24 h in this study. Af- ter the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylaiton status in the promoter region of RASSF1A gene was re- versed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expres- sion at transcriptional level and a 40 kDa (1kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The ex- perimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Effect of antisense DNMT3b gene eukaryotic expression plasmid on expression of the DNMT3b gene in human biliary tract carcinoma cells

BACKGROUND: Hypermethylation of the promoter region is one of the major mechanisms of tumor suppressor gene inactivation. DNA methyltransferase 3b (DNMT3b), an enzyme that participates in the establishment of de novo methylation patterns, is highly expressed in many tumor cells and tissues, and it is closely associated with hypermethylation of the promoter of tumor suppressor genes. The aim of this study was to explore the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the expression of the DNMT3b gene in human biliary tract carcinoma cell. METHODS: The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into the human biliary tract carcinoma cell line QBC-939 with lipofectamine transfection reagent, and positive cell clones were formed using G418 selection after transfection. The constructed recombinant plasmid was transfected into QBC-939 cells successfully and was confirmed by amplification of the exogenous neo^R gene with the polymerase chain reaction method. The expression of DNMT3b gene mRNA and protein was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry respectively. RESULTS: Following transfection, the mRNA level of the DNMT3b gene decreased from 0.956±0.053 to 0.209±0.023, and the protein level of the DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. Very significant differences were observed both at the transcription and posttranscription levels in the expression of the DNMT3b gene between the non-tranfection group and the antisense DN- MT3b gene eukaryotic expression plasmid transfection group (P<0.01). CONCLUSIONS: Transfection with the antisense DNMT3b gene eukaryotic expression plasmid can significantly reduce the expression level of the DNMT3b gene in the human biliary tract carcinoma cell line QBC-939. This study may provide a valid method to investigate the function of the DNMT3b gene and its role in biliary tract carcinoma.

Effect of Antisense MBD1 Gene Eukaryotic Expression Plasmid on Expression of MBD1 Gene in Human Biliary Tract Carcinoma Cells

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912±0.022 to 0. 215±0. 017, and the protein level of MBD1 gene also decreased from （80.19±5.05） %to （35.11±4.05） %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group （P〈0.01）. It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.

Expression Analysis of Aquaporin-1 (Aqp-1) in Human Biliary Tract Carcinoma

Background: Aquaporins (AQPs) are important in controlling bile water secretion. AQP is related to the invasion and metastasis of cancer. However, the relationship of biliary tract cancer is not clear. The role of AQP-1 in cancer cell is also unknown. Methhods: We analyzed AQP-1 expression using tissue microarray (TMA) in 99 samples immunohistochemically (50 gallbladder carcinoma, 39 bile duct carcinoma and 10 Papilla Vater carcinoma patients who underwent surgery at our department from 1997 to 2011). Gene expressions were evaluated by the combination of the immunohistological intensity and distribution. The expression level is compared to the clinico-pathological data of the patients. Results: In the TMA, depth of tumor invasion and histological type are associated with AQP-1 expression. The group of patients with high AQP-1 expression is associated with higher rates of disease specific survival (log-rank p = 0.013). Cox’s proportional hazard model reveals that AQP-1 expression is an independent prognostic factor (RR, 0.324;p = 0.001) in multivariate analysis. There is a correlation between AQP-1 expression and tumor invasion. Conclusions: These observations of this study suggest that AQP-1 expression may be favorable biomarkers associated with prognosis and tumor invasion in biliary tract carcinoma.

The relationship between loss expression of DPC4/Smad4 gene and carcinogenesis of pancreatobiliary carcinoma

Objective: To clarify the relationship between loss of DPCA gene expression and pathogenesis of pancreato- biliary carcinoma. Methods: 75 slides of normal duct (20), hyperplasia (15), dysplasia (15), invasive carcinoma (25) from patients with pancreatic diseases including pancreatic carcinoma (25 patients), chronic pancreatitis (6), pancreas injury (2) and 71 slides of common bile duct (CBD) carcinoma (38), gallbladder carcinoma (18), hilar bile duct (HBD) carcinoma (15) from patients with primary biliary tract carcinoma were analyzed for the expression of DPC4 protein by im- munohistochemical staining. Results: All specimens from 20 cases of normal duct and 15 cases of hyperplasia showed marked expres- sion of DPC4 protein. The frequency of loss expres- sion of the DPC4 gene was 33 % in dysplasia, and 48% in invasive carcinoma. There was a significant statistical difference between byperplasia and dyspla- sia (P<0.01) and in dysplasia vs invasive carcinoma (P<0.05). The frequency of loss expression of the DPC4 gene was 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, and 13% in HBD carcinoma. The frequency of loss expression of the DPCA gene was significantly different in CBD carcinoma vs gall- bladder carcinoma and HBD carcinoma (P<0.01). Conclusions: Inactivation of the DPC4 gene occurs late in the neoplastic progression of pancreatic carci- noma. The frequency of DPC4 gene alternation was different in various locations of biliary tract carcino- ma. In CBD carcinoma, this frequency is similar to that in pancreatic carcinoma, indicating their similar molecular alternations.

Current status of minimally invasive liver surgery for cancers

Hepatocellular carcinoma(HCC)patients have chronic liver disease with functional deterioration and multicentric oncogenicity.Liver surgeries for the patients should be planned on both oncological effects and sparing liver function.In colorectal patients with post-chemotherapy liver injury and multiple bilateral tumors,handling multiple tumors in a fragile/easy-to-bleed liver is an important issue.Liver surgery for biliary tract cancers is often performed as a resection of large-volume functioning liver with extensive lymphadenectomy and bile duct resection/reconstruction.Minimally invasive liver surgery(MILS)for HCC is applied with the advantages of laparoscopic for cases of cirrhosis or repeat resections.Small anatomical resections using the Glissonian,indocyanine greenguided,and hepatic vein-guided approaches are under discussion.In many cases of colorectal liver metastases,MILS is applied combined with chemotherapy owing to its advantage of better hemostasis.Two-stage hepatectomy and indocyanine green-guided tumor identification for multiple bilateral tumors are under discussion.In the case of biliary tract cancers,MILS with extensive lymphadenectomy and bile duct resection/reconstruction are developing.A robotassisted procedure for dissection of major vessels and handling fragile livers may have advantages,and well-simulated robot-assisted procedure may decrease the difficulty for biliary tract cancers.