Biogenesis and function of microRNAs can be influenced by genetic variants in the pri-miRNA sequences leading to phenotypic variability. This study aims to identify single nucleotide polymorphisms (SNPs) affecting the expression levels of bone-related mature microRNAs and thus, triggering an osteoporotic phenotype. An association analysis of SNPs located in pri-miRNA sequences with bone mineral density (BMD) was performed in the OSTEOMED2 cohort (n = 2183). Functional studies were performed for assessing the role of BMD-associated miRNAs in bone cells. Two SNPs, rs6430498 in the miR-3679 and rs12512664 in the miR-4274, were significantly associated with femoral neck BMD. Further, we measured these BMD-associated microRNAs in trabecular bone from osteoporotic hip fractures comparing to non-osteoporotic bone by qPCR. Both microRNAs were found overexpressed in fractured bone. Increased matrix mineralization was observed after miR-3679-3p inhibition in human osteoblastic cells. Finally, genotypes of rs6430498 and rs12512664 were correlated with expression levels of miR-3679 and miR-4274, respectively, in osteoblasts. In both cases, the allele that generated higher microRNA expression levels was associated with lower BMD values. In conclusion, two osteoblast-expressed microRNAs, miR-3679 and miR-4274, were associated with BMD; their overexpression could contribute to the osteoporotic phenotype. These findings open new areas for the study of bone disorders.

Conclusions Methods ResultsSNP rs6430498 in the miR-3679 and rs12512664 in the miR-4274 were significantly associated with FN BMD (Table 4). The A alleles for rs6430498 (minority allele) and rs12512664 (majority allele) were found to be associated with lower BMD values. ObjectivesFunding Disclosures miRNAs have been extensively studied in bone research, particularly their relationship to osteoporosis (De-Ugarte et al, BMC Med Genomics, 2015; Seeliger et al, J Bone Min Research, 2014) . However, the miRNA expression signatures described in patients with osteoporosis do not provide evidence of causality because the altered pattern could be a consequence of the disease or even unrelated to the pathogenesis. Another approach in miRNAs studies is the association analysis between one SNP within a candidate miRNA (miR-SNP) or in a miRNA target site, and one disease related-outcome. In this case, the associated variant is likely involved in the pathophysiology or confers susceptibility to develop the disease (Estrada et al, Nat Genet, 2012).The aims of this study were (1) to identify SNPs within candidate miRNAs in order to perform an association study with bone mineral density (BMD), the main outcome used to define osteoporosis and (2) to validate in bone cells this miR-SNP association with the osteoporotic phenotype.SNPs located in pre-miRNA sequences that (1) bind to the mRNA 3UTR of genes related with bone metabolism or (2) found to be highly expressed in human osteoblasts were selected. Validated SNPs with a MAF>0.01 (n=5) were genotyped in the OSTEOMED2 cohort (Table 1) to assess their association with LS BMD and FN BMD (Table 3). Multivariate linear regression models were fitted to assess the association between genotyped SNPs and BMD. Potential confounders considered for adjustment were densitometer devices, body mass index (BMI) and age. MiRNAs which harbored BMD-associated SNPs were quantified by qPCR in order to compare the expression levels between OP and non-OP bone samples (Table 2). Mann-Whitney U test was performed for OP and non-OP group comparisons. Human primary osteoblasts were cultured for DNA and RNA extraction and sorted by genotype for both rs6430498 and rs12512664. The correlation between expression levels of mature miRNA miR-3679-3p and miR-4274 with its corresponding genotypes was analyzed using linear regression. All analyses were two-tailed, and p-values<0.05 were considered significant.l Genetic variants in miR-3679 and miR-4274 are associated with femoral neck bone mineral density. In both cases, alleles associated with lower BMD correlated with higher expression levels of respective mature miRNAs in human osteoblastic cells which were found overexpressed in fractured bone samples.l Our results open new exploratory avenues for future studies in the bone field and the treatment of osteoporosis.The authors state that they have no conflict of interest. This work was supported by the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) and FEDER funds.