AIM:To identify methylation profile and novel tumor marker of extrahepatic cholangiocarcinoma(CCA)with high throughout microarray.METHODS:Differential methylation profile was compared between normal bile duct epitheli...AIM:To identify methylation profile and novel tumor marker of extrahepatic cholangiocarcinoma(CCA)with high throughout microarray.METHODS:Differential methylation profile was compared between normal bile duct epithelial cell lines and CCA cell lines by methyl-DNA immunoprecipitation (MeDIP)microarray.Bisulfite-polymerase chain reaction (BSP)was performed to identify the methylated allels of target genes.Expression of target genes was investigated before and after the treatment with DNA demethylating agent.Expression of candidate genes was also evaluated by immunofluorescence in 30 specimens of CCA tissues and 9 normal bile duct tissues.RESULTS:Methylation profile of CCA was identified with MeDIP microarray in the respects of different gene functions and signaling pathways.Interestingly,97 genes with hypermethylated CpG islands in the promoter region were homeobox genes.The top 5 hypermethylated homeobox 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’-aza2’-deoxycytidine.Significant expression differences were found between normal bile duct and extrahepatic CCA tissues(66.67%-100%vs 3.33%-10%).CONCLUSION:HOXA2,HOXA5,HOXA11,HOXB4 and HOXD13 may work as differential epigenetic biomarkers between malignant and benign biliary tissues.展开更多
Background CCAAT/enhancer-binding protein β（C/EBPβ） is required for mitotic clonal expansion （MCE） during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3...Background CCAAT/enhancer-binding protein β（C/EBPβ） is required for mitotic clonal expansion （MCE） during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPβ is required for preadipocyte proliferation, and identify new target genes of C/EBPP with chromatin immunoprecipitation （ChlP）-on-chip. Methods Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. Chip was performed at 20 hours after induction with specific anti-C/EBPβ antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.Results A total of 110 candidate genes were identified. BTG3 associated nuclear protein （SMAR1, Banp） and tripartite motif-containing 35 （Hls5, trim35） were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBP3 to the promoter of banp and trim35 was verified by ChlP-PCR. Conclusion C/EBPβ may regulate preadipocyte proliferation through activation of banp and trim35.展开更多
基金Supported by The grant for"Development of Novel Nano-Drug Delivery System Loaded with Traditional Chinese Anticancer Medicine for the Targeted Therapy of Malignant Tumors"issued by the Chinese Ministry of Science and Technology,Grant No. 2010DFA31870
文摘AIM:To identify methylation profile and novel tumor marker of extrahepatic cholangiocarcinoma(CCA)with high throughout microarray.METHODS:Differential methylation profile was compared between normal bile duct epithelial cell lines and CCA cell lines by methyl-DNA immunoprecipitation (MeDIP)microarray.Bisulfite-polymerase chain reaction (BSP)was performed to identify the methylated allels of target genes.Expression of target genes was investigated before and after the treatment with DNA demethylating agent.Expression of candidate genes was also evaluated by immunofluorescence in 30 specimens of CCA tissues and 9 normal bile duct tissues.RESULTS:Methylation profile of CCA was identified with MeDIP microarray in the respects of different gene functions and signaling pathways.Interestingly,97 genes with hypermethylated CpG islands in the promoter region were homeobox genes.The top 5 hypermethylated homeobox 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’-aza2’-deoxycytidine.Significant expression differences were found between normal bile duct and extrahepatic CCA tissues(66.67%-100%vs 3.33%-10%).CONCLUSION:HOXA2,HOXA5,HOXA11,HOXB4 and HOXD13 may work as differential epigenetic biomarkers between malignant and benign biliary tissues.
基金This work was supported by the National Natural Science Foundation of China （No. 30700403）.
文摘Background CCAAT/enhancer-binding protein β（C/EBPβ） is required for mitotic clonal expansion （MCE） during adipogenesis. It is still unclear how C/EBPp regulates MCE in the earlier differentiation programs of 3T3-L1 preadipocytes. The purpose of this paper was to understand why C/EBPβ is required for preadipocyte proliferation, and identify new target genes of C/EBPP with chromatin immunoprecipitation （ChlP）-on-chip. Methods Postconfluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation using a standard differentiation protocol. Chip was performed at 20 hours after induction with specific anti-C/EBPβ antibodies. The precipitated DNA was amplified, labeled and hybridized with a mouse promoter microarray. Compared with the control in which the ChIP experiment was performed with non-specific antibody, only the genes with a signal increasing more than 2 fold were considered as candidate genes.Results A total of 110 candidate genes were identified. BTG3 associated nuclear protein （SMAR1, Banp） and tripartite motif-containing 35 （Hls5, trim35） were two target genes among the 110 candidate genes which are involved in cell cycle regulation; the binding of C/EBP3 to the promoter of banp and trim35 was verified by ChlP-PCR. Conclusion C/EBPβ may regulate preadipocyte proliferation through activation of banp and trim35.
