IRF4 suppresses osteogenic differentiation of BM-MSCs by transcriptionally activating miR-636/DOCK9 axis

Zhang, Xuepu; Zhang, Yue; Yang, Limin; Wu, Yuexin; Ma, Xiaohu; Tong, Gang; Ban, Zhaoliang; Zhao, Haosen

doi:10.1016/j.clinsp.2022.100019

Abstract

Objectives

Osteoblasts are derived from Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), which play an indispensable role in bone formation. In this study, the authors aim to investigate the role of IRF4 in the osteogenic differentiation of BM-MSCs and its potential molecular mechanism.

Methods

The authors used lentivirus infection to overexpress IRF4 in BM-MSCs. The expression of IRF4 and osteogenesis-related genes were detected by qRT-PCR and western blot analysis. The osteogenic differentiation of BM-MSCs was evaluated by Alkaline Phosphatase (ALP) activity, Alizarin red staining, and Alkaline Phosphatase (ALP) staining. Chromatin Immunoprecipitation (ChIP), Dual-Luciferase reporter assay and RNA Immunoprecipitation Assay were applied to confirm the regulatory mechanism between IRF4, miR-636 and DOCK9.

Results

The authors found IRF4 was down-regulated during the osteogenic differentiation of BM-MSCs, and IRF4 overexpression could decrease the osteogenic differentiation of BM-MSCs by specifically promoting the reduction of Alkaline Phosphatase (ALP) activity and down-regulating osteogenic indicators, including OCN, OPN, Runx2 and CollA1. Mechanistically, IRF4 activated microRNA-636 (miR-636) expression via binding to its promoter region, and Dedicator of Cytokinesis 9 (DOCK9) was identified as the target of miR-636 in BM-MSCs. Moreover, the damage in the capacity of osteogenic differentiation of BM-MSCs induced by IRF4 overexpression could be rescued by miR-636 inhibition.

Conclusions

In summary, this paper proposed that IRF4/miR-636/DOCK9 may be considered as targets for the treatment of osteoporosis (OP).

Keywords
Osteoporosis; BM-MSCs; Osteogenic differentiation; miR-636; DOCK9; ALP

[1] Authors' contributions

Xuepu Zhang, Yue Zhang and Haosen Zhao conceived the study and designed the experiments. Limin Yang, Yuexin Wu and Xiaohu Ma contributed to the data collection. Gang Tong and Zhaoliang Ban performed the data analysis and interpreted the results. Xuepu Zhang and Yue Zhang wrote the manuscript. Haosen Zhao contributed to the critical revision of the article. All authors read and approved the final manuscript.