title: Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta.
abstract: MicroRNA (miRNA) are non-coding small RNA that regulate gene expression. MiR-328 is reported to influence breast cancer resistance protein (BCRP) expression in cancer cells. As a large inter-individual difference in BCRP levels is observed in various human tissues, the contribution of miR-328 to these differences is of interest. We hypothesized that DNA methylation in the miR-328 promoter region is responsible for the difference in miR-328 levels, leading to inter-individual variability in BCRP levels in human placenta. The association between placental miR-328 and BCRP levels was analyzed, and then DNA methylation in the miR-328 5'-flanking region and regulatory mechanisms causing inter-individual differences in miR-328 and BCRP levels were examined. MiR-328 expression was significantly correlated with BCRP mRNA (Rs = -0.560, P < 0.01) and protein (Rs = -0.730, P < 0.01) levels. It was also up-regulated by the demethylating agent 5-aza-2'-deoxycytidine in BCRP-expressing cells. Luciferase assays with differentially methylated reporter constructs indicated that methylation in the miR-328 5'-flanking region including a predicted CpG island remarkably decreased transcriptional activity compared to that in unmethylated constructs. We selected CCAAT/enhancer binding protein α (C/EBPα), located within the predicted CpG island, by in silico analysis. To elucidate the role of C/EBPα in miR-328 expression, a chromatin immunoprecipitation assay, promoter deletion analysis, and electrophoretic mobility shift assay (EMSA) were performed. C/EBPα-binding site-truncated constructs showed significantly decreased promoter activity, and EMSA indicated that the C/EBPα-binding sites were located in the CpG island. Finally, the methylation patterns of several CpG dinucleotides proximal to two C/EBPα-binding sites in the miR-328 5'-flanking region were correlated negatively with miR-328 levels, and positively with BCRP levels in human placental samples. These results suggest that methylation patterns in the miR-328 5'-flanking region are involved in the inter-individual difference in BCRP levels in human placenta.
authors.affiliation: Department of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Jumpei
authors.initials: J
authors.lastname: Saito
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hirota
authors.forename: Shinji
authors.initials: S
authors.lastname: Furuta
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kobayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takane
authors.forename: Ichiro
authors.initials: I
authors.lastname: Ieiri
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D014158:Transcription, Genetic
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C548170:MIRN328 microRNA, human; D035683:MicroRNAs; D009363:Neoplasm Proteins