Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta.
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 the...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3749162?pdf=render |
| _version_ | 1818155805497622528 |
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| author | Jumpei Saito Takeshi Hirota Shinji Furuta Daisuke Kobayashi Hiroshi Takane Ichiro Ieiri |
| author_facet | Jumpei Saito Takeshi Hirota Shinji Furuta Daisuke Kobayashi Hiroshi Takane Ichiro Ieiri |
| author_sort | Jumpei Saito |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T14:48:14Z |
| format | Article |
| id | doaj.art-15adc9a1018f48ecbc9d308565786e1f |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T14:48:14Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj.art-15adc9a1018f48ecbc9d308565786e1f2022-12-22T01:01:35ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0188e7290610.1371/journal.pone.0072906Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta.Jumpei SaitoTakeshi HirotaShinji FurutaDaisuke KobayashiHiroshi TakaneIchiro IeiriMicroRNA (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.http://europepmc.org/articles/PMC3749162?pdf=render |
| spellingShingle | Jumpei Saito Takeshi Hirota Shinji Furuta Daisuke Kobayashi Hiroshi Takane Ichiro Ieiri Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta. PLoS ONE |
| 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. |
| title_full | Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta. |
| title_fullStr | Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta. |
| title_full_unstemmed | Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta. |
| title_short | Association between DNA methylation in the miR-328 5'-flanking region and inter-individual differences in miR-328 and BCRP expression in human placenta. |
| title_sort | association between dna methylation in the mir 328 5 flanking region and inter individual differences in mir 328 and bcrp expression in human placenta |
| url | http://europepmc.org/articles/PMC3749162?pdf=render |
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