MiR-494-3p knockdown promotes osteogenic differentiation of bone marrow mesenchymal stem cells in ovariectomised rats by targeting Sirt1/TLR4/NF-κB pathway

Introduction: Osteoporosis is a bone disease characterised by low bone mass, deterioration of bone tissue, and disruption of bone microarchitecture. MicroRNAs have been found to play an important role in osteoporosis. MicroRNA (miR)-494 is inhibited during bone angiogenesis, and its overexpression reduces osteogenic differentiation gene expression. Sirtuin 1 (SIRT1) is a histone deacetylase with multiple cellular activities including increasing bone mass and delaying the onset of osteoporosis. MiR-494-3p has been predicted in computer-assisted bioinformatics analysis to target the 3'UTR of SIRT1 mRNA. The aim of the present study was to assess the effect of miR-494-3p on ovariectomy (OVX)-induced osteoporosis in rats and the relevant mechanisms.

Material and methods: Osteoporosis in female rats was induced by OVX, and bone microarchitectural changes were evaluated by means of microCT. MiR-494-3p and SIRT1 expression in femurs were evaluated by RT-qPCR or Western blotting. Bone marrow mesenchymal stem cells (BMSCs) were isolated from rat femurs and identified by flow cytometry. Then, BMSCs were transfected with miR-494-3p inhibitor/mimic, si-Sirt1 and negative controls as well as pcDNA3.1-TLR4 and empty pcDNA3.1 vector. Osteogenic cell differentiation was assessed via Alizarin Red, Alkaline Phosphatase (ALP) and Oil Red O staining.

Results: MiR-494-3p level was upregulated, and SIRT1 mRNA and protein levels were downregulated, in femurs of OVX rats. Functionally, miR-494-3p inhibited osteogenic differentiation of cultured rat BMSCs. Mechanistically, miR-494-3p regulated the SIRT1 3'UTR to activate TLR4/NF-κB signalling, and SIRT1 inhibition and TLR4 overexpression reversed the enhancing effect of miR-494-3p knockdown on osteogenic differentiation.

Conclusions: MiR-494-3p represses osteogenic differentiation of BMSCs in OVX rats through SIRT1/TLR4/NF-κB signalling.

Sirt1; TLR4/NF-κB; miR-494-3p; osteogenic differentiation; osteoporosis; ovariectomy.