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.
Highlights
Breast cancer resistance protein (BCRP) is constitutively expressed in normal human tissues including the intestine, liver, blood–brain barrier, breast, and placenta, as well as tumor tissues, and is involved in multidrug resistance because BCRP acts as an efflux transporter of anti-cancer drugs [1]
It was reported that some single nucleotide polymorphisms (SNPs) (i.e., C421A, G34A and C376T genotypes) significantly impacted on BCRP function [2,3,4]
A single CpG island in the miR-328 5’-flanking region We focused on DNA methylation in the miR-328 5’-flanking region
Summary
Breast cancer resistance protein (BCRP) is constitutively expressed in normal human tissues including the intestine, liver, blood–brain barrier, breast, and placenta, as well as tumor tissues, and is involved in multidrug resistance because BCRP acts as an efflux transporter of anti-cancer drugs [1]. Several non-synonymous single nucleotide polymorphisms (SNPs) in the BCRP gene (ABCG2) have been associated with reduced BCRP transport activity [2,3,4,5,6,7]. Based on these observations, the genetic variation in BCRP is considered to contribute to the inter-individual variability in the pharmacokinetics of BCRP substrate drugs. Studies have showed that miR-328 is important in regulating BCRP expression, but little is known about its contribution to the variability in BCRP levels among individuals. We focused on the inter-individual variability in BCRP and miR-328 levels in the human placenta. We evaluated the importance of methylation patterns to individual miR-328 levels
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