Crosstalk between microRNA-21–5p and the transcription factor Dec1 maintains osteoblast function

Abstract

MicroRNAs are associated with pivotal post-transcriptional gene regulation in bone formation. Human differentiated embryonic chondrocyte expressed gene 1 (Dec1) is also involved in regulating osteoblastogenesis. In the present study, we aimed to investigate the distinctive role of miR-21–5p and Dec1 in osteoblast function and to determine their biological functions. MC3T3-E1 pre-osteoblastic cells were used for in vitro analyses. miR-21–5p knockout (KO) mice, Dec1KO mice and age-matched wild-type (WT) mice were used to characterize the influence of miR-21–5p and Dec1 deficiencies on bone formation. Morphological analyses [micro-computed tomography (micro-CT)] were performed, and measurements were collected to validate miR-21-5pKO mice. Histopathological changes in mouse femur tissues were assessed by H-E staining, Azan staining, Masson's Trichrome staining, and Toluidine Blue staining. Quantitative real-time RT-PCR, western blotting and immunohistochemical staining were used to characterize the expression levels of Alkaline Phosphatase, Runx2, Osterix, Osteopontin, Dec1 and miR-21–5p. Bioinformatics analyses and dual-luciferase reporter assays were performed to confirm Dec1 as a target of miR-21–5p. Dec1 expression was gradually increased from day 7 of osteoblast induction, while miR-21–5p showed a peak at day 21. In non-induced osteoblasts, a mechanistically gain-of-function transfection study with a miR-21–5p mimic enhanced Runx2 and Osterix expression but suppressed Dec1. miR-21-5pKO mice had reduced bone growth. Dec1-deficient mice showed advanced bone formation at the age of 12 weeks compared to WT mice. The Dec1 deficiency upregulated Runx2 and Osterix expression in Dec1KO mouse femurs. Those changes, however, were reversed in miR-21-5pKO mouse femurs compared to WT mouse femurs. Dual-luciferase reporter assays showed that Dec1 is a possible downstream target of miR-21–5p. These findings showed that the reduced osteogenic potential due to a miR-21–5p deficiency is achieved by enhanced Dec1 expression and that the miR-21–5p/Dec1 axis is involved in regulating osteoblast function.