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Sodium taurocholate cotransporting polypeptide (NTCP) is not only an important transporter for bile acid absorption into the liver, but also a functional receptor for HBV and HDV, and extensive studies have been performed for its structure, function, gene characteristics, and expression and regulation mechanisms. NTCP is also associated with chronic viral hepatitis, nonalcoholic fatty liver disease, liver fibrosis, primary biliary cholangitis, and hepatocellular carcinoma. This article elaborates on the role of NTCP in various hepatobiliary diseases, so as to provide new direction for the diagnosis and treatment of related diseases.
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Hepatocellular carcinoma (HCC) is a type of tumor with a high incidence rate, a low rate of early diagnosis, and poor prognosis, and its development and progression involve many factors. As an important organelle in cells, mitochondria is the "energy factory" of cells and is one of the main sites for the production of reactive oxygen species in vivo, and it also participates in the regulation of cell apoptosis. There are varying degrees of changes in mitochondrial membrane, oxidation respiratory chain, mitochondrial dynamics, mitochondrial DNA, and mitochondrial calcium homeostasis during the development and progression of HCC, and such changes may affect the progression of HCC. This article systematically elaborates on the association between mitochondria and HCC, so as to provide a new direction for the diagnosis and treatment of HCC.
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Cholangiocarcinoma (CCA) is a category of highly heterogeneous and aggressive malignancy mainly originating from bile duct epithelial cells. The median survival time of untreated CCA patients is approximately 12?24 months, and the effectiveness and durability of surgical resection and neoadjuvant chemotherapy are limited. Results of the next-generation sequencing show that dysregulation of the immune system plays an important role in the pathogenesis of CCA. It has opened up new possibilities for the study of therapies targeting the natural course of aggressive CCA, such as immune checkpoint inhibitors, adoptive cell therapy, and tumor vaccines. Based on the current status of immunotherapy for CCA, the authors review the efficacy and dilemmas of current CCA immunotherapy strategies and look forward to the future treatment prospects of CCA.
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Mirrizi syndrome is a rare benign disease of biliary tract. With the continuous emergence of new concepts and technologies in the field of disease diagnosis and treatment, the standardization of diagnosis and treatment for biliary diseases is also updated. Mirrizi syndrome is difficult to be diagnosed pre-operatively, and intraoperative unclear Calot′s triangle and tissue adhesion caused by gallbladder inflammation and gallbladder fistula formation increase the risk of biliary tract injury, which lead certain challenges for its surgical treatment. Therefore, strict preoperative evaluation and selection of corresponding surgical procedure for different classification of Mirrizi syndrome are of great significance for reducing postoperative complications. In addition, the development of laparoscopic technology reduces the surgical trauma and enhances recovery, but puts forward higher requirements for the experience and operation of surgeons. It is important that reasonably select laparoscopic surgery indications, correctly hold indications of bilioenetric anastomosis, and prevent iatrogenic injury of bile duct on the premise of clearing stones, which is the basic strategy for surgical treatment of Mirrizi syndrome. Based on relevant literatures and clinical practice, the author investigate the therapeutic principles and strategies of Mirrizi syndrome.
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Immunoglobulin G (IgG)4-related pancreatitis, also named as type Ⅰ autoimmune pancreatitis, is recognized as a pancreatic manifestation of IgG4-related disease. It is very hard to differentiate it from pancreatic cancer due to similar clinical manifestations with pancreatic cancer. Diagnosis of IgG4-related pancreatitis mainly relies on pathological examination and imaging examination. Corticosteroids are the main treatment for IgG4-related pancreatitis, and the risk of relapse is still high with steroid taper or following withdrawal of steroids. Based on researches on IgG4-related pancreatitis and our clinical experience, the authors have analyzed and summarized pathogenesis, clinical manifestations, clinical diagnosis, differentiation from pancreatic cancer, clinical treatment of IgG4-related pancreatitis, and suggested that elucidation of its mechanism and induction of immunotherapy may improve its efficacy.
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Immunoglobulin G (IgG) 4-related pancreatitis,also named as type I autoimmune pancreatitis,is recognized as a pancreatic manifestation of IgG4-related disease.It is very hard to differentiate it from pancreatic cancer due to similar clinical manifestations with pancreatic cancer.Diagnosis of IgG4related pancreatitis mainly relies on pathological examination and imaging examination.Corticosteroids are the main treatment for IgG4-related pancreatitis,and the risk of relapse is still high with steroid taper or following withdrawal of steroids.Based on researches on IgG4-related pancreatitis and our clinical experience,the authors have analyzed and summarized pathogenesis,clinical manifestations,clinical diagnosis,differentiation from pancreatic cancer,clinical treatment of IgG4-related pancreatitis,and suggested that elucidation of its mechanism and induction of immunotherapy may improve its efficacy.
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<p><b>OBJECTIVE</b>To investigate the expression of miR-29b in cholangiocarcinoma and explore its effects on cell proliferation and apoptosis of cholangiocarcinoma cells.</p><p><b>METHODS</b>Real-time PCR was used to detect the expression of miR-29b in cholangiocarcinoma cells line QBC939 and cholangiocarcinoma tissues. The lentiviral vector LV-hsa-miR-29b and blank vector were constructed to infect QBC939 cells. MTT assay and cell clone formation assay were performed to assess the changes in the cell proliferation and clone formation, respectively; flow cytometry was employed to evaluate the effect of miR-29b overexpression on cell cycle and apoptosis.</p><p><b>RESULTS</b>The expression of miR-29b was significantly down-regulated in QBC939 cells and cholangiocarcinoma tissues as compared with H-69 cells and normal tissues ( < 0.01). Compared with the blank vector, the lentiviral vector LV-hsa-miR-29b caused significantly increased expression of miR-29b in QBC939 cells ( < 0.01), which exhibited suppressed cell proliferation and clone formation ( < 0.01 or 0.05), cell cycle arrest at the S phase ( < 0.05), and significantly increased cell apoptosis ( < 0.01).</p><p><b>CONCLUSIONS</b>As a tumor-suppressing miRNA, miR-29b is down-regulated in cholangiocarcinoma, and its overexpression can suppress the proliferation and induce apoptosis of cholangiocarcinoma cells.</p>
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Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.
Subject(s)
Apoptosis , Biliary Tract Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Cholangiocarcinoma/metabolism , DNA (Cytosine-5-)-Methyltransferases/biosynthesis , DNA (Cytosine-5-)-Methyltransferases/genetics , Down-Regulation , Gene Expression Regulation, Neoplastic , Genetic Vectors , Mice, Nude , Neoplasm TransplantationABSTRACT
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 micromol/L for 24 h in this study. After 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, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=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 experimental 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.
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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. After 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 reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression 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 experimental 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.
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<p><b>OBJECTIVE</b>To investigate the effects of mutant exogenous P27(kip1) gene on chemosensitivity of human cholangiocarcinoma cell line.</p><p><b>METHODS</b>The recombinant vector was constructed and the mutant P27(kip1) gene was transfected into human cholangiocarcinoma cell line (QBC(939)). RT-PCR and Western blot were used to determine the expression of target genes. The effects of 5-fluorouracil (5-FU), mitomycin C (MMC) and cyclophosphamide (CTX) on the transfected cells were detected by assaying the apoptotic rate and growth inhibition by methabenzthiazuron (MTT) assay and flow cytometry (FCM).</p><p><b>RESULTS</b>The mutant exogenous P27(kip1) gene was expressed effectively in the cells, and the expression enhanced the apoptosis and growth inhibition of QBC(939) inducted by 5-FU, MMC and CTX. The ratio of growth inhibiting increased significantly from 41.89% (5-FU), 45.59% (MMC), 38.91% (CTX) to 56.15% (5-FU), 55.65% (MMC), 51.69% (CTX), and apoptosis index from 13.76% +/- 3.03% (5-FU), 11.76% +/- 3.99% (MMC), 10.46% +/- 2.10% (CTX) to 41.39% +/- 4.32% (5-FU), 35.94% +/- 2.71% (MMC), 34.46% +/- 2.32% (CTX) (P < 0.05).</p><p><b>CONCLUSIONS</b>The exogenous P27(kip1) gene transfer can remarkably increase the drug sensibility of the cholangiocarcinoma cells. The strategy targeted to control the cell cycle may be more effective in cancer treatment by combination of P27(kip1) gene therapy.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Antineoplastic Agents , Pharmacology , Apoptosis , Bile Duct Neoplasms , Pathology , Bile Ducts, Intrahepatic , Cell Division , Cell Line, Tumor , Cholangiocarcinoma , Pathology , Cyclin-Dependent Kinase Inhibitor p27 , Genetics , Pharmacology , Drug Synergism , Genetic Vectors , TransfectionABSTRACT
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.
Subject(s)
Biliary Tract Neoplasms/metabolism , Biliary Tract Neoplasms/pathology , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Eukaryotic Cells/metabolism , Gene Expression Regulation, Neoplastic , Genetic Vectors , Oligonucleotides, Antisense/genetics , Plasmids/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , TransfectionABSTRACT
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 MBD1gene 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 eukary otic 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.
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Objective To study the effect of transfection of antisense MBD1 gene eukaryotic expression vector on the expression of MBD1 gene in human cholangiocarcinoma cell line QBC-939.Methods The(constructed) antisense MBD1 gene eukaryotic expression vector was transfected into the human(cholangiocarcinoma) cell line QBC-939 using lipofectamine transfection reagents,and positive cell clones were obtained using G418 selection after transfection.The constructed recombinant vector was transfected into(QBC-939) cells successfully and was confirmed by amplifying the exogenous neo~R gene with PCR method.The expression level of MBD1 gene mRNA and protein was detected by RT-PCR and FCM methods respectively.Results Following the transfection,the MBD1 gene mRNA level in human cholangiocarcinoma cell line QBC-939 decreased from 0.912?0.022 to 0.215?0.017,and the MBD1 gene protein level also(decreased) from(80.19?5.05)% to(35.11?4.05)%.There were very significant differences on the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression vector transfection group(P
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Objective To construct eukaryotic expression vector of antisense MBD1 gene fragment and to provide a tool for studying MBD1 gene function. Methods PCR primers were designed according to the coding sequence of MBD1 gene. Xba I and Kpn I recognition sequences and cutting sites were added to the 5' end of the sense and antisense primer respectively. The 342 bp specific PCR fragment was obtained from the cDNA of biliary tract carcinoma cell line QBC-939 using RT-PCR, the purified PCR fragment was then inserted reversely into the multiple cloning site of eukaryotic expression vector pcDNA3. 1 ( + ). The constructed recombinant plasmid was identified by PCR confirmation, Xba I and Kpn I double enzyme digestion and DNA sequencing. Results The 322 bp specific DNA band was obtained by PCR, Xba I and Kpn I double digestion produced a 327 bp and a 5. 4 kb DNA band which represent the inserted target gene fragment and the vector respectively. The sequencing result confirmed that the sequence of inserted fragment was correct. Conclusion The eukaryotic expression vector of antisense MBD1 gene fragment was constructed successfully by using gene cloning technique. It will be a useful tool for studying MBD1 gene function in DNA methylation and tumorigenesis.
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Objective To investigate the effects of the tumor suppressor gene PTEN in growing inhibition and down-regulating mTOR in cholangiocarcinoma QBC939 cells in vitro.Methods QBC939 cells were transfected with plasmids wild-type PTEN and C124S-PTEN in vitro.After transfection,the expression of the PTEN and phosphorylation of AKT and mTOR was detected by Western blot.Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results Compared with the control,the expression of phosphorylation AKT was decreased and mTOR were down-regulated respectively when transfected with the wild-type PTEN.However,after transfection with mutation-type PTEN,the level of PTEN in the cells by increased,but phosphorylation AKT level and mTOR expression had no significant change.Conclusions PTEN can be actived by phosphorylated AKT.Actived AKT decreased the mTOR which led to tumor cells apoptosis and regulation of the tumor cell cycle.In the pathway of signal transmission of PI3K/AKT/PTEN/mTOR,PTEN and mTOR are closely related through phosphorylation of AKT.
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Objective To study the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the growth of human cholangiocarcinoma cell line QBC939,and to explore the role of DNMT3b in the cholangiocarcinoma tumorigenesis.Methods The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into QBC939 cells using liposome.The expression level of DNMT3b protein was detected by Western blot after stable transfection.The growth curves of transfected cells and un-transfected cells were observed by MTT method respectively.The cell proliferation ability was also observed by the test of colony formation in soft agar.The alterations of the cell cycle and the apoptosis rate were detected by FCM.Results Following the transfection,the protein level of DNMT3b decreased significantly;transfection with antisense DNMT3b gene eukaryotic expression plasmid did not affect the cell growth curve of QBC939,and did not decrease the cell colony formation rate(P=0.717);transfection with antisense DNMT3b gene also did not result in cell cycle alterations or induce cell apoptosis(P=0.089).Conclusions Transfection with antisense DNMT3b gene eukaryotic expression plasmid can down-regulate the expression level of DNMT3b in QBC939.It can not affect the growth and proliferation of human cholangiocarcinoma cell line QBC939,nor alter the cell cycle and induce cell apoptosis.