AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined...AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.展开更多
基金Supported by National Natural Science Foundation of China,No. 81071998Hubei Natural Science Foundation,No.2008CDB159Specialized Research Fund for the Doctoral Program of Higher Education,No. 20070487114
文摘AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.
