Identification Of Methylation Profile Of Cholangiocarcinoma

PurposeOur aims were to investigate the methylation profile of cholangiocarcinoma to screen methylation biomarker of cholangiocarcinoma by high-throughout MeDIP microarray, establish 3D model of human cholangiocarcinoma epithelial cell line TFK-1 and identify the differential effect of hydralazine and valproic acid between 2D and 3D cultured models.Methods1. In an effort to identify new cancer-specific methylation markers, we employed a high-throughput MeDIP microarray chip to explore the methylation profile differences between TFK-1 and BEC and utilized and NimbleScanTM2.2 and SignaMap software to analyze the methylation level and function of interesting genes. Validation of methylation level of candidate genes has been performed by bisulfite sequence-PCR. Furthermore, expression of target genes was investigated after the treatment with DNA demethylation agent. Expression of candidate genes was also observed by immunofluorescence in 30 chlangiocarcinoma tissues and 9 normal bile duct tissues.2.3D cultured cells were established in culture plates coated with poly-HEMA on a gyratory shaker.Viability of 2D and 3D cultured cells was examined by WST-1 viability assay and FDA/PI staining. Methylation status of promoters regarding 3 tumor suppressor genes APC, E-Cadherin, and HOXA5 was investigated by methylation-specific PCR.3. Apoptosis was detected by mixed dye including both Annexin-FITC and Propidium with flow cytometry technique, and changes of methylation and transcription of mRNA were explored by RT-PCR and MSP techniques after the intervention of hydralazine and valproic acid either alone or combined for 24 hours and 48 hours.Results1. Compared to BEC cell line,2103 CpG islands were hypermethylation, including 1672 known genes and 531 unknown genes, and 970 hypermethylated CpG islands located in the promoter area, including 317 unknown genes and 653 known genes. Interestingly,94 genes with hypermethylated CpG islands in promoter region were Homeobox genes. The top 5 hypermethylated genes validated by BSP were HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%). Expression of these genes was reactivated with 5'-aza-2'-deoxycytidine. Significant expression differences were detectable between normal bile duct and cholangiocarcinoma tissues (66.67-100% normal vs 3.33%-10% cancer).2. The average diameters of TFK-1 spheroids were in the range of 350-400μm. WST-1 results demonstrated that TFK-1 spheroid cells were more resistant to the epigenetic drugs and 11.22fold higher IC 50 values of hydralazine and valproic acid than did the same cells growing in monolayer culture. FDA/PI staining indicated that death rate was presented in dose-dependent behavior. Higher dose of epigenetic drugs were needed to reverse hypermethylation status in 3D cells compared to 2D cells, while parallel dose-dependent characteristic existing in 2D cells didn't present in 3D cells.3. The transcription o f mRNA of TMS1/ASC gene and caspase-1 re-expressed again after the combined intervention o f hydralazine and valproic acid, which was higher than that of the cells treated with either hydralazine or valproic acid alone (P<0.05). The demethylation effect of 48 h by combind intervention treatment was better than that o f 24 h (P<0.05). The growth of the QBC 939 cell line was inhibited, and flow cytometry showed marked increase of apoptosis (49.88±0.044)%. Conclusion1. According to the results of data on MeDIP-chip (NimbleScan v2.5; Roche-NimbleGen), differentially methylated DNA concentrated on the Calcium, MAPK, Wnt, Hedgehog, TGF-beta signaling pathway and are involved in growth of cells, DNA synthesis and repair, cell differentiation,cell apoptosis, cell transcription factor, cell migration and adhesion and angiogenesis.2.94 genes with hypermethylated CpG islands in promoter region were Homeobox genes. Demonstrated that HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%) expressed in normal but low expressed in cholangiocarcinoma, which provided us a new strategies to discover new biomarker of cholangiocarcinoma.3. The unique characteristics of spheroid culture, affecting the consequences of epigenetic therapy, are more complex in 3D spheroid cells than that of monolayer culture. Higher dose of epigenetic drugs were needed to reverse hypermethylation status in 3D cells compared to 2D cells, while parallel dose-dependent characteristic existing in 2D cells didn't present in 3D cells. We speculated that the novel structural changes between cells from 2D monolayer to 3D spheroid culture might require re-expression of cell-cell adhesion genes to maintain the formation of 3D spheroids.4. TMS1 /ASC gene and caspase-1 may re-express after the synergistical intervention of hydralazine and valproic acid, and the effect is more obvious as the treatment time is extended. The apoptosis of TFK-1 cell line is increased, which may be indued by caspase-1 passway. PartⅠIdentification of methylation profile of cholangiocarcinoma with MeDIP microarrayPurpose Cholangiocarcinoma is a malignant cancer arising from the neoplastic transformation of cholangiocytes. It has been identified that methylation events will change the gene expression patterns without causing the changes in the nucleotide sequence of the genetic code, which is a main mechanism of tumorigenesis. The object of this study is to explore methylation profile differences between human cholangiocarcinoma cell line TFK-1 and biliary epithelial cell line BEC.Methods In an effort to identify new cancer-specific methylation markers, we employed a high-throughput MeDIP microarray chip to explore the methylation profile differences between TFK-1 and BEC and utilized and NimbleScanTM2.2 and SignaMap software to analyze the methylation level and function of interesting genes.Results Compared to BEC cell line,2103 CpG islands were hypermethylation, including 1672 known genes and 531 unknown genes, and 970 hypermethylated CpG islands located in the promoter area, including 317 unknown genes and 653 known genes which contains 11 HOX genes.Conclusion By using MeDIP screen we identified multiple hypermethylated genes involving cell cycle, differentiation, adhesion and metastasis, and tumor angiogenesis, which distribute to the mechanism of tumor. These data suggested new different methylated genes may work as new target genes for the search of possible specific cholangiocarcinoma marker. PartⅡEffects of DNA methylation on Hox gene expression in cholangiocarcinomaPurpose Homeobox genes are members of a transcription factor family and play a crucial role in embryonic development and in the control of cell differentiation proliferation, apoptosis and signaling path. Numerous examples of aberrant Hox gene expression induced by hypermethylation of promoter region have been found in cancer. The purpose was to investigate effect of DNA methylation on HOX expression in cholangiocarcinoma.Methods Validation of methylation level of candidate genes has been performed by bisulfite sequence-PCR. Furthermore, expression of target genes was investigated after the treatment with DNA demethylation agent. Expression of candidate genes was also observed by immunofluorescence in 30 chlangiocarcinoma tissues and 9 normal bile duct tissues.Results We identified methylation profile of cholangiocarcinoma with MeDIP microarray, relating to different gene functions and signaling pathways. Interestingly,94 genes with hypermethylated CpG islands in promoter region were Homeobox genes. The top 5 hypermethylated genes validated by BSP were HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%). Expression of these genes was reactivated with 5'-aza-2'-deoxycytidine. Significant expression differences were detectable between normal bile duct and cholangiocarcinoma tissues (66.67-100% normal vs 3.33%-10% cancer).Conclusion Our research demonstrated methylation profile of cholangiocarcinoma for the first time with high throughput MeDIP chips. These findings supported that HOXA2, HOXA5, HOXA11, HOXAB4 and HOXD13 may work as targets for diagnosis screening and therapeutic intervention. PartⅢComparison of the effect of epigenetic therapy in 2D and 3D cholangiocarcinoma modelsPurpose Epigenetic modifications affect gene expression pattern of cholangiocarcinoma, but very little data of epigenetic therapy of cholangiocarcinoma exists in vivo. Since multicellular tumor spheroids can mimic biological characteristics of tumor,3D spheroid model have been widely applied in systemic drug screening. Our aims were to establish 3D model of human cholangiocarcinoma epithelial cell line TFK-1 and identify the differential effect of epigenetic drugs between 2D and 3D cultured models.Methods 3D cultured cells were established in culture plates coated with poly-HEMA on a gyratory shaker.Viability of 2D and 3D cultured cells was examined by WST-1 viability assay and FDA/PI staining. Methylation status of promoters regarding 3 tumor suppressor genes APC, E-Cadherin, and HOXA5 was investigated by methylation-specific PCR.Results The average diameters of TFK-1 spheroids were in the range of 350-400μm. WST-1 results demonstrated that TFK-1 spheroid cells were more resistant to the epigenetic drugs and 11.22fold higher IC 50 values of hydralazine and valproic acid than did the same cells growing in monolayer culture. FDA/PI staining indicated that death rate was presented in dose-dependent behavior. Higher dose of epigenetic drugs were needed to reverse hypermethylation status in 3D cells compared to 2D cells, while parallel dose-dependent characteristic existing in 2D cells didn't present in 3D cells.Conclusions The unique characteristics of spheroid culture, affecting the consequences of epigenetic therapy, are more complex in 3D spheroid cells than that of monolayer culture and 3D spheroid is a promising model for epigenetic therapy.PartⅣEffects of hydralazine and valproic acid on the methylation of ASC /TMS1 of human cholangiocarcinoma cell linePurpose To investigate changes of methylation status of ASC/TMS1 in QBC 939 cell line of cholangiocarcinoma before and after combined DNA methylation and histone deacetylase inhibitors treatment, and the correlation of the apoptosis which is induced by caspase-1, and methylation status of ASC/TMS1.Methods Apoptosis was detected by mixed dye including both Annexin-FITC and Propidium with flow cytometry technique, and changes of methylation and transcription of mRNA were explored by RT-PCR and MSP techniques after the intervention of hydralazine and valproic acid either alone or combined for 24 hours and 48 hours.Results The transcription o f mRNA of TMS1/ASC gene and caspase-1 re-expressed again after the combined intervention o f hydralazine and valproic acid, which was higher than that of the cells treated with either hydralazine or valproic acid alone (P<0.05). The demethylation effect of 48 h by combind intervention treatment was better than that o f 24 h (P<0.05). The growth of the QBC 939 cell line was inhibited, and flow cytometry showed marked increase of apoptosis (49.88±0.044)%.Conclusions TMS1 /ASC gene and caspase-1 may re-express after the synergistical intervention of hydralazine and valproic acid, and the effect is more obvious as the treatment time is extended. The apoptosis of TFK-1 cell line is increased, which may be indued by caspase-1 passway.