Abstract
MicroRNAs (miRs) play an essential role in the regulation of bone formation and homeostasis. miR-185 has been reported to negatively regulate osteogenesis in vitro. However, whether it has an impact on in vivo bone homeostasis remains unknown. Here, we demonstrated that primary osteoblasts and mesenchymal stem cells derived from miR-185-knockout (KO) mice exhibited enhanced osteogenesis. Further, we constructed an ovariectomized mouse model to investigate the role of miR-185 during osteoporosis. Micro-computed tomography revealed an increased bone volume in KO compared to wild-type mice 6 weeks after surgery, indicating redundant bone formation after miR-185 depletion. Dual-luciferase reporter assays identified biglycan (Bgn), which promotes bone formation through the BMP/Smad pathway, as the direct target of miR-185. Taken together, these findings indicate that blocking miR-185 expression increases bone formation during osteoporosis, which may partly occur through the regulation of Bgn expression and BMP/Smad signaling.
Highlights
Bone homeostasis is a dynamic balance that includes bone formation by osteoblasts and bone resorption by osteoclasts
An increasing body of evidence suggests that miR-185 is a key regulator in bone homeostasis
MiR-185 modulated the expression of tumor growth factor (TGF-β1) after ankle fracture within 2 weeks, indicating a role in fracture recovery in patients[14]
Summary
Antibodies and reagents Antibodies to GAPDH (Sungene Biotech, KM9002), Alp (Abcam, ab108337), osterix (Osx) (Bioss, bs-1110R), Dlx[2] (Proteintech, 26244-1-AP), Bgn (Proteintech, 16409-1AP), Bmp[2] (Proteintech, 18933-1-AP), Smad[1] (Proteintech, 10429-1-AP), and phospho-Smad1/5/8 (CST,13820) were purchased commercially. All the mice were humanely euthanized, and bone samples were harvested and fixed. The medium was changed every 3 days throughout the experiments and cells were harvested at indicated time points. MiRNA mimic and small interfering RNA (siRNA) transfection was performed using Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Real-time PCR TRIzol reagent (Invitrogen Life Technologies, 15596026) was used to extract total RNA from cells and tissues. Alkaline phosphatase and Alizarin Red S staining and activity determination A total of 2 × 104 cells were seeded per 24-well plate, and cultured in OIM for 7 or 14 days. Alkaline phosphatase (ALP) staining buffer were prepared according to the manufacturer's instructions (CWBIO, cw0051), and added to the cells.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
