Down-regulated microRNA-199a-3p enhances osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Kdm3a in ovariectomized rats

Abstract

Osteoporosis is a prevalent systemic skeletal disorder entailing bone fragility and increased fracture risk, often emerging in post-menopausal life. Emerging evidence implicates the dysregulation of microRNAs (miRNAs or miRs) in the progression of osteoporosis. This study investigated the effect of miR-199a-3p on osteoporosis and its underlying mechanism. We first examplished an ovariectomized (OVX)-induced rat osteoporosis model, and then isolated mesenchymal stem cells (MSCs) from bone marrow of the model rats. The overexpression and knock down of miR-199a-3p were conducted in OVX rats and MSCs to verify the role of miR-199a-3p on MSC differentiation. Calcium nodules were measured using alizarin red S (ARS) staining. RT-qPCR and Western blot assay were performed to measure the expression of miR-199a-3p, Kdm3a and osteogenic differentiation-related markers in rat tissues and cells. The correlation between miR-199a-3p and Kdm3a was confirmed using dual-luciferase reporter assay. The enrichment of Kdm3a at the Erk2 and Klf2 promoter was assessed using chromatin immunoprecipitation (ChIP) assay. Isolated MSCs were positive for CD29, CD44, CD90, and CD45, suggesting successful isolation of MSCs. There was increased expression of miR-199a-3p and inhibited osteogenic differentiation in OVX rats. Kdm3a was negatively targeted by miR-199a-3p. Our results also demonstrated that Kdm3a elevated the expression of Erk2 and Erk2 by promoting Erk2 and Klf2 demethylation, which further contributed to osteogenic differentiation. Overall, our results revealed a regulatory network of miR-199a-3p in osteogenic differentiation, highlighting miR-199a-3p as a potential target for therapeutic interventions in osteoporosis.

Effect of Anti-Osteoporotic Treatments on Circulating and Bone MicroRNA Patterns in Osteopenic ZDF Rats

“…High levels of miR-199a-3p were also reported in exosomes from human bone marrow MSCs [ 14 ], and the authors suggested that the transport of miR-199a-3p in MSC exosomes is a component of intercellular communication in the bone niche. When the decrease in bone mass and the increase in body weight shown by ZDF rats ( Figure 1 ) is taken into consideration, this downregulation of miR-199a-3p in ZDF rats is in contrast with reports showing that the enhanced expression of miR-199a-3p promoted adipogenesis from bone-marrow-derived MSCs [ 15 ], while its downregulation enhanced the osteogenic differentiation of bone marrow MSCs in ovariectomized rats [ 16 ]. However, supporting our data, miR-199a-3p was upregulated in osteocytic areas in ovariectomized (OVX) mice and was also an inductor of catabolic bone cell autophagy in osteocyte-like MLO-Y4 cells [ 17 ].…”

Exploring the relationship between systemic lupus erythematosus and osteoporosis based on bioinformatics

“…54 Wu et al have demonstrated that miR-199a-3p can reduce the expression of alkaline phosphatase, Runx2, Osx, and other osteogenic differentiation factors by inhibiting Erk2 phosphorylation, leading to the acceleration of bone loss and the occurrence of OP. 55 Wang et al have demonstrated that miR-29b expression in PBMC of SLE patients is upregulated; AKT kinase is abnormally activated; anti-dsDNA titer is upregulated, and C3 level is downregulated. 56 This is contrary to the expression shown in studies of OP: the expression of miR-29b is significantly downregulated in the bone marrow of OP patients, reducing the activation of AKT kinase, and thereby inhibiting stem cell osteogenic differentiation.…”

BMSC-derived exosomal miR-27a-3p and miR-196b-5p regulate bone remodeling in ovariectomized rats

“…The rats were anesthetized by an intraperitoneal injection of 2% 2,2,2-tribromoethanol (Macklin, Shanghai, China). Then, bilateral ovaries were removed from the OVX rats, while peripheral ovarian fat was removed from the Sham rats, as described previously ( Wu et al, 2021 ). After completion of operation the resuscitated rats were kept in the same conditions as before operation.…”

“…Flow cytometric characterization was performed as described previously ( Wu et al, 2021 ). Briefly, the passage 3 BMSCs were digested with 0.25% trypsin and suspended to a concentration of 1 × 10 6 cells/mL with cell staining buffer (BioLegend, San Diego, CA, USA).…”