MicroRNAs Differentially Regulate Carbonyl Reductase 1 (CBR1) Gene Expression Dependent on the Allele Status of the Common Polymorphic Variant rs9024

Abstract

MicroRNAs (miRNAs) are small RNAs responsible for the post-transcriptional regulation of a variety of human genes. To date, their involvement in the regulation of CBR1 is unknown. This study reports for the first time the identification of microRNA-574-5p (hsa-miR-574-5p) and microRNA-921 (hsa-miR-921) as two miRNAs capable of interacting with the 3′-untranslated region (3′-UTR) of the CBR1 gene and downregulating CBR1 expression. Furthermore, we demonstrate that a common single-nucleotide polymorphism (SNP) in the CBR1 3′-UTR (rs9024, CBR1 1096G>A) differentially impacts the regulation of CBR1 by hsa-miR-574-5p and hsa-miR-921 dependent on genotype. First, four candidate miRNAs were selected based on bioinformatic analyses, and were tested in Chinese hamster ovary (CHO) cells transfected with CBR1 3′-UTR constructs harboring either the G or A allele for rs9024. We found that hsa-miR-574-5p and hsa-miR-921 significantly decreased luciferase activity in CHO cells transfected with the CBR1 3′-UTR construct carrying the major rs9024 G allele by 35% and 46%, respectively. The influence of these miRNAs was different in cells transfected with a CBR1 3′-UTR construct containing the minor rs9024 A allele in that only hsa-miR-574-5p had a demonstrable effect (i.e., 52% decrease in lucifersase activity). To further determine the functional effects of miRNA-mediated regulation of polymorphic CBR1, we assessed CBR1 protein expression and CBR1 enzymatic activity for the prototypical substrate menadione in human lymphoblastoid cell lines with distinct rs9024 genotypes. We found that hsa-miR-574-5p and hsa-miR-921 significantly decreased CBR1 protein (48% and 40%, respectively) and CBR1 menadione activity (54% and 18%, respectively) in lymphoblastoid cells homozygous for the major rs9024 G allele. In contrast, only hsa-miR-574-5p decreased CBR1 protein and CBR1 activity in cells homozygous for the minor rs9024 A allele, and did so by 49% and 56%, respectively. These results suggest that regulation of human CBR1 expression by hsa-miR-574-5p and hsa-miR-921 depends upon rs9024 genotype status.