A GDF15 3′ UTR variant, rs1054564, results in allele-specific translational repression of GDF15 by hsa-miR-1233-3p

Growth differentiation factor 15 (GDF15) is a strong predictor of cardiovascular events and mortality in individuals with or without cardiovascular diseases. Single nucleotide polymorphisms (SNPs) in microRNA (miRNA) target sites, also known as miRSNPs, are known to enhance or weaken miRNA-mRNA interactions and have been linked to diseases such as cardiovascular disease and cancer. In this study, we aimed to elucidate the functional significance of the miRSNP rs1054564 in regulating GDF15 levels. Two rs1054564-containing binding sites for hsa-miR-873-5p and hsa-miR-1233-3p were identified in the 3′ untranslated region (UTR) of the GDF15 transcript using bioinformatics tools. Their activities were further characterized by in vitro reporter assays. Bioinformatics prediction suggested that miRNA binding sites harboring the rs1054564-G allele had lower free energies than those with the C allele and therefore were better targets with higher affinities for both hsa-miR-873-5p and hsa-miR-1233-3p. Reporter assays showed that luciferase activity was significantly decreased by rs1054564-G-containing 3′ UTRs for both miRNAs (P < 0.05) and was restored by miRNA inhibitors. Comparing the fold suppression of the two miRNAs, only that of hsa-miR-1233-3p showed significant changes between the rs1054564-G- and C-containing 3′ UTRs (P = 0.034). In addition, western blots showed that transfection of both miRNA mimics significantly decreased endogenous GDF15 expression in a melanoma cell line (P < 0.05). Taken together, our findings demonstrate that GDF15 is a target of hsa-miR-873-5p and hsa-miR-1233-3p and that the rs1054564-C allele partially abolishes hsa-miR-1233-3p-mediated translational suppression of GDF15. These results suggest that rs1054564 confers allele-specific translational repression of GDF15 via hsa-miR-1233-3p. Our work thus provides biological insight into the previously reported clinical association between rs1054564 and plasma GDF15 levels.