miR-765 inhibits the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting BMP6 via regulating the BMP6/Smad1/5/9 signaling pathway

Abstract

Background: The process of bone repair is heavily dependent on the ability of human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation. MicroRNAs have been shown to regulate this osteogenic process. This study aimed to investigate the role of miR-765 in the osteogenic differentiation of hMSCs. Methods: We transfected hMSCs with lentiviral constructs to knock down or overexpress this miRNA, allowing us to assess its role in osteogenesis via assessing the expression of the relevant markers alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2), and osteocalcin (OCN), with further functional measurements made via quantifying ALP activity and conducting Alizarin Red S staining. The targeting of the 3′-untranslated region (UTR) of BMP6 by miR-765 was examined via luciferase assay. We used hMSCs with altered miR-765 expression to assess p-Smad1/5/9 levels via Western blotting over the course of osteogenic differentiation. We also assessed the osteogenic differentiation of hMSCs in which miR-765 was knocked down and at the same time as a BMP/Smad signaling inhibitor was added to disrupt Smad1/5/9 phosphorylation. Results: We found miR-765 overexpression to inhibit osteogenesis-associated gene upregulation during osteogenic differentiation of hMSCs, whereas knockdown of this miRNA was associated with increased expression of these genes. Using luciferase reporter assays, we confirmed direct miR-765 binding to the 3′-untranslated region (UTR) of BMP6. We also found that miR-765 overexpression reduced Smad1/5/9 phosphorylation, and knockdown of this miRNA enhanced this phosphorylation on BMP6/Smad1/5/9 signaling. The osteogenic differentiation of hMSCs in which miR-765 had been knocked down was further weakened upon the addition of a BMP/Smad signaling inhibitor relative to miR-765 knockdown alone. Conclusions: Together, these results thus suggest that miR-765 is able to inhibit hMSC osteogenic differentiation by targeting BMP6 via regulation of the BMP6/Smad1/5/9 signaling pathway. Our findings may offer molecular insights of value for the development of novel therapeutic treatments for bone diseases including osteoporosis.

Roles of MicroRNAs in Bone Destruction of Rheumatoid Arthritis

“…The expression of Smad7 was up-regulated and the levels of p-Smad2 and p-Smad3 were decreased by inhibiting miR-877-3p (He G. et al, 2019). Additionally, by regulating the translation and expression of BMP pathway related factors, miR-144-3p (Huang C. et al, 2016), miR-217 (Zhu et al, 2017), miR-92a (Yan et al, 2018), and miR-765 (Wang T. et al, 2020) also inhibited osteoblast differentiation.…”

MicroRNA‑142 protects MC3T3‑E1 cells against high glucose‑induced apoptosis by targeting β‑catenin

“…Research has highlighted the roles of miRs as the critical mediators of osteogenic development. miR-765 inhibited the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting BMP6 (15). miR-214 may protect MC3T3-E1 cells against apoptosis by targeting ATF4 and ameliorating oxidative stress (16).…”

The Role of MicroRNAs in Bone Metabolism and Disease

“…The overexpression of miR-155 in MSCs was reported to decrease the expression of Runx2, OPN, phosphorylated Smad1/5/8 (p-Smad1/5/8), and OCN and to reduce osteogenesis in vitro as well as in vivo by targeting Runx2 and bone morphogenetic protein receptor 9 [ 61 ]. Similarly, miR-765 overexpression in hMSCs also inhibited osteogenic-gene expression as well as phosphorylation of Smad1/5/9 through BMP6 and subsequently osteogenic differentiation of hMSC [ 62 ]. Bone morphogenetic protein type Ib receptor BMPR1b is a serine/threonine kinase receptor that plays a vital role in MSC osteogenic differentiation.…”