AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically ...AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham’s F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics. RESULTS: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods.CONCLUSION: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.展开更多
AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined ...AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a noncancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR)mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS: The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro , suggesting its potential as a therapeutic target.展开更多
BACKGROUND In the past decades,the potential of microRNA(miRNA)in cancer diagnostics and prognostics has gained a lot of interests.In this study,a meta-analysis was conducted upon the pooled miRNA microarray data of c...BACKGROUND In the past decades,the potential of microRNA(miRNA)in cancer diagnostics and prognostics has gained a lot of interests.In this study,a meta-analysis was conducted upon the pooled miRNA microarray data of cholangiocarcinoma(CCA).AIM To identify differentially expressed(DE)miRNAs and perform functional analyses in order to gain insights to understanding miRNA-target interactions involved in tumorigenesis pathways of CCA.METHODS Raw data from 8 CCA miRNA microarray datasets,consisting of 443 samples in total,were integrated and statistically analyzed to identify DE miRNAs via comparison of levels of miRNA expression between CCA and normal bile duct samples using t-tests(P<0.001).The 10-fold cross validation was performed in order to increase the robustness of the t-test results.Our data showed 70 up-regulated and 48 down-regulated miRNAs in CCA. GeneOntology and pathway enrichment analyses revealed that mRNA targets of DEmiRNAs were significantly involved in several biological processes. The mostprominent dysregulated pathways included phosphatidylinositol-3 kinases/Akt,mitogen-activated protein kinase and Ras signaling pathways.CONCLUSIONDE miRNAs found in our meta-analysis revealed dysregulation in major cancerpathways involved in the development of CCA. These results indicated thenecessity of understanding the miRNA-target interactions and the significance ofdysregulated miRNAs in terms of diagnostics and prognostics of cancers.展开更多
基金Supported by Thailand Research Fund (The Royal Golden Jubilee Ph.D. Program) and Rajavithi Hospital Fund
文摘AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham’s F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics. RESULTS: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods.CONCLUSION: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.
基金Supported by Grant from Thailand Research Fund and Faculty of Science, Mahidol University, Thailand (to Suthiphongchai T)Institutional Strengthening Program,Faculty of Science,Mahidol University (to Thummarati P)
文摘AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a noncancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR)mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS: The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro , suggesting its potential as a therapeutic target.
基金Supported by the Thailand Research Fund,No.DBG5980006UK-Thailand Research Collaborations(Newton Fund),No.MR/N01247X/1.
文摘BACKGROUND In the past decades,the potential of microRNA(miRNA)in cancer diagnostics and prognostics has gained a lot of interests.In this study,a meta-analysis was conducted upon the pooled miRNA microarray data of cholangiocarcinoma(CCA).AIM To identify differentially expressed(DE)miRNAs and perform functional analyses in order to gain insights to understanding miRNA-target interactions involved in tumorigenesis pathways of CCA.METHODS Raw data from 8 CCA miRNA microarray datasets,consisting of 443 samples in total,were integrated and statistically analyzed to identify DE miRNAs via comparison of levels of miRNA expression between CCA and normal bile duct samples using t-tests(P<0.001).The 10-fold cross validation was performed in order to increase the robustness of the t-test results.Our data showed 70 up-regulated and 48 down-regulated miRNAs in CCA. GeneOntology and pathway enrichment analyses revealed that mRNA targets of DEmiRNAs were significantly involved in several biological processes. The mostprominent dysregulated pathways included phosphatidylinositol-3 kinases/Akt,mitogen-activated protein kinase and Ras signaling pathways.CONCLUSIONDE miRNAs found in our meta-analysis revealed dysregulation in major cancerpathways involved in the development of CCA. These results indicated thenecessity of understanding the miRNA-target interactions and the significance ofdysregulated miRNAs in terms of diagnostics and prognostics of cancers.
