Association between DNA Methylation in the miR-328 5'-Flanking Region and Inter-individual Differences in miR-328 and BCRP Expression in Human Placenta

Jumpei Saito, Takeshi Hirota, Shinji Furuta, Daisuke Kobayashi, Hiroshi Takane, Ichiro Ieiri

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish
Article numbere72906
JournalPloS one
Volume8
Issue number8
DOIs
Publication statusPublished - Aug 21 2013

Fingerprint

5' Flanking Region
DNA methylation
DNA Methylation
placenta
Individuality
breast neoplasms
Placenta
protein synthesis
CCAAT-Enhancer-Binding Proteins
Breast Neoplasms
binding proteins
Proteins
proteins
protein binding
CpG Islands
Methylation
Assays
methylation
Protein Binding
binding sites

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Association between DNA Methylation in the miR-328 5'-Flanking Region and Inter-individual Differences in miR-328 and BCRP Expression in Human Placenta. / Saito, Jumpei; Hirota, Takeshi; Furuta, Shinji; Kobayashi, Daisuke; Takane, Hiroshi; Ieiri, Ichiro.

In: PloS one, Vol. 8, No. 8, e72906, 21.08.2013.

Research output: Contribution to journalArticle

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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.",
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AU - Saito, Jumpei

AU - Hirota, Takeshi

AU - Furuta, Shinji

AU - Kobayashi, Daisuke

AU - Takane, Hiroshi

AU - Ieiri, Ichiro

PY - 2013/8/21

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