MiR-296 Promotes Osteoblast Differentiation by Upregulating Cbfal

Abstract

We aim to identify the potential role of miR-296, which is a class of endogenous non-coding RNAs in regulating osteoblast differentiation. Human osteoblast cell line, hFOB 1.19 cells, were transfected with miR-296 overexpression or inhibition plasmid to upregulate or downregulate miR-296 expression, and then co-transfected with anti-Cbfal antibody to knockdown Cbfal. Alizarin red staining, alkaline phosphatase (ALP) activity, and enzyme-linked immunosorbent assay assays were performed to evaluate the extent of osteoblast differentiation. MTT assays were applied to measure cell proliferation. Wound healing and transwell assays were used to determine the ability of osteoblast migration and invasion. Flow cytometry assay was used to measure cell apoptosis and cell cycle change. The mRNA and protein expression of Cbfal, OSX, and Col-1 were confirmed by RT-qPCR and western blot respectively. We found that miR-296 overexpression contributed to a significant enhancement of matrix mineralization, ALP activity, and osteocalcin expression comparing with the negative control, whereas knockdown of miR-296 caused an opposite result. Meanwhile, the treatment of miR-296 promotes osteoblast migration, invasion, and proliferation, while it inhibits cell apoptosis. Compared with the negative control groups, the cells transfected with miR-296 also presented a much higher expression of Cbfal, OSX, and Col-1. Further experiments confirmed that the positive regulatory role of miR-296 in osteoblast differentiation is achieved by upregulating Cbfal expression. In conclusion, miR-296 promotes osteoblast differentiation by upregulating Cbfal expression in hFOB 1.19 cells, which may be used as a potential therapeutic target for the treatment of bone diseases in future.

lncRNA FGD5-AS1 Regulates Bone Marrow Stem Cell Proliferation and Apoptosis by Affecting miR-296-5p/STAT3 Axis in Steroid-Induced Osteonecrosis of the Femoral Head

“…Here, miR-296-5p weakened the protective effect of FGD5-AS1 in steroid-induced ONFH, indicating that miR-296-5p upregulation can inhibit cell proliferation and differentiation and promote apoptosis in Dex-treated hBMSCs. On the contrary, Yu et al proposed that miR-296 promoted osteoblast differentiation by upregulating Cbfal [ 24 ]. The reason for this difference may be that the cells we selected and the target genes of miR-296 are different.…”

miRNAs Related to Different Processes of Fracture Healing: An Integrative Overview

“…MiR-296 also promoted osteoblast differentiation, though no exact mechanism of action has been identified yet. Overexpression of miR-296 prevented apoptosis of osteoblasts, whilst enhancing matrix mineralization, as well as increased expression levels of osteogenic marker genes RUNX2, OCN, COL1A1, and ALP (104). Similar to miR-194, miR-302a stimulated osteoblastic differentiation by inhibiting COUP-TFII, thereby promoting RUNX2 activity.…”

MicroRNA Regulation of Bone Marrow Mesenchymal Stem Cells in the Development of Osteoporosis in Obesity

“…An increase in miR-296 expression can trigger the osteogenic differentiation of MSCs (osteoblastogenesis) and decrease the activity of the transcription factor Pten, which is critical for BMSC differentiation into adipocytes [82] (Table 2, Ref. [82][83][84][85][86][87][88][89]). In a human osteoblast cell line (hFOB 1.19 cells), the overexpression of miR-296 contributed to a significant increase in matrix mineralization, ALP activity, osteocalcin expression, and increased corebinding factor al (Cbfal) expression [83] (Table 2).…”

“…[82][83][84][85][86][87][88][89]). In a human osteoblast cell line (hFOB 1.19 cells), the overexpression of miR-296 contributed to a significant increase in matrix mineralization, ALP activity, osteocalcin expression, and increased corebinding factor al (Cbfal) expression [83] (Table 2).…”