The Vitamin D Receptor Regulates MiR-140-5p and Targets the MAPK Pathway in Bone Development

Background: Skeletal development is a complicated process. The status of vitamin D (VD) is closely related to fetal bone development in the embryonic period. Recently, miRNAs have been found to participate in the regulation of skeletal growth and development in several species. However, the mechanisms underlying the interactions among vitamin D, its receptor (VDR), and miRNAs during the process of bone development remain unclear. The aim of this study was to identify miRNAs that are regulated by 1,25(OH)D in murine osteoblasts and to analyze the relationship and the effects of VD/VDR and miRNAs in vitro and in vivo.

Methods: We performed miRNA sequencing in murine primary osteoblasts and in an osteoblast cell line treated with 1,25(OH)D to identify miRNAs in these cells. After qRT-PCR validation, miR-140-5p was selected for further analysis. We assessed the pathways comprising predicted target genes for several expressed miRNAs, including miR-140-5p, validated predicted target genes in the MAPK pathway by qRT-PCR, and explored the correlation between VD/VDR and miR-140-5p in vitro and in vivo.

Results: 88 miRNAs in murine primary osteoblasts and 49 miRNAs in osteoblast cell line were found to be differentially expressed. MiR-140-5p was upregulated in these 2 types of murine osteoblasts. The expression of miR-140-5p was promoted by 1,25(OH)D through transcriptional activation by VDR, with targeted inhibition of MAPK signaling in osteoblasts. A positive correlation between vitamin D/VDR and miR-140-5p was observed in VDR-knockout mice and in 165 human serum specimens. These data show for the first time that VDR transcriptionally activates miR-140-5p. Therefore, the VD/VDR/miR-140-5p/MAPK signaling axis plays an important role in transmitting the effects of 1,25(OH)D.

Conclusion: Our results demonstrate a novel regulatory mechanism by which miR-140-5p targets the MAPK pathway by means of VD/VDR in vitro and in vivo. These findings provide a new reference for mechanistic research and therapeutic approaches for vitamin D-related bone diseases.