Role of hsa-mirna-223-3p in regulating osteogenic differentiation of human bone marrow mesenchymal stem cells

Geng YAO

ABSTRACT

BACKGROUND:

Studies have found that miR-223-3p is highly expressed in osteoporosis patients, but the related mechanism is rarely studied.

OBJECTIVE:

To investigate the regulatory effect of hsa-miR-223-3p on osteogenic differentiation of bone marrow mesenchymal stem cells.

METHODS:

Bone marrow mesenchymal stem cells were isolated and obtained by Percoll density gradient centrifugation. The cells were induced into osteogenic differentiation, and overexpressed hsa-miR-223-3p. RT-qPCR and western blot assay were used to detect the changes in the mRNA and protein levels of Wnt signaling pathway markers Wnt5a and its downstream signaling molecules OPG and RUNX2. PmirGLO/Wnt5a-3'UTR and pmirGLO/Wnt5a-3'UTR mut plasmids were constructed and co-transfected with hsa-miR-223-3p mimics/control plasmid into 293T cells to verify the targeting relationship between hsa-miR-223-3p and Wnt5a by dual luciferase reporter gene system. RESULTS AND

CONCLUSION:

(1) With the prolongation of osteogenic induction time, the level of alkaline phosphatase in cell culture fluid gradually increased. The deposition of dark red or reddish brown calcium salt nodules was gradually increased according to alizarin red staining results. (2) Overexpression of hsa-miR-223-3p decreased the mRNA and protein levels of Wnt5a, OPG and RUNX2 in cells. (3) hsa-miR-223-3p directly targeted the 3'UTR region of Wnt5a and reduced the luciferase activity of Wnt5a. This targeting effect disappeared when the binding site of Wnt5a was mutated. Blocking hsa-miR-223-3p increased the luciferase activity of wild-type Wnt5a, while the luciferase activity of mutant Wnt5a remained unchanged. (4) The results showed that during the differentiation of bone marrow mesenchymal stem cells into osteoblasts, overexpression of hsa-miR-223-3p could affect the expression of Wnt signaling pathway marker Wnt5a and its downstream signaling molecules OPG and RUNX2. hsa-miR-223-3p could directly bind to Wnt5a, which was negatively regulated as a downstream target gene of hsa-miR-223-3p.