Knockdown of FOXA1 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly via activation of the ERK1/2 signalling pathway

Stem Cell Research & Therapy - Tập 13 Số 1
Lijun Li1,2,3,4, Yibo Wang1,2,3,4, Zhongxiang Wang1,2,3,4, Deting Xue1,2,3,4, Chao Dai1,2,3,4, Xiang Gao1,2,3,4, Jianfei Ma5, Kai Hang1,2,3,4, Zhijun Pan1,2,3,4
1Clinical Research Center of Motor System Disease of Zhejiang Province, Hangzhou City, People’s Republic of China
2Department of Orthopedics Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou City, People’s Republic of China
3Key Laboratory of Motor System Disease Research and Precision Therapy of Zhejiang Province, Hangzhou City, People’s Republic of China
4Orthopedics Research Institute of Zhejiang University, Hangzhou City, People’s Republic of China
5Key Laboratory of Image Information Processing and Intelligent Control, School of Artificial Intelligence and Automation, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

Tóm tắt

Abstract Background The available therapeutic options for large bone defects remain extremely limited, requiring new strategies to accelerate bone healing. Genetically modified bone mesenchymal stem cells (BMSCs) with enhanced osteogenic capacity are recognised as one of the most promising treatments for bone defects. Methods We performed differential expression analysis of miRNAs between human BMSCs (hBMSCs) and human dental pulp stem cells (hDPSCs) to identify osteogenic differentiation-related microRNAs (miRNAs). Furthermore, we identified shared osteogenic differentiation-related miRNAs and constructed an miRNA-transcription network. The Forkhead box protein A1 (FOXA1) knockdown strategy with a lentiviral vector was used to explore the role of FOXA1 in the osteogenic differentiation of MSCs. Cell Counting Kit-8 was used to determine the effect of the knockdown of FOXA1 on hBMSC proliferation; real-time quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to investigate target genes and proteins; and alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS) were used to assess ALP activity and mineral deposition, respectively. Finally, a mouse model of femoral defects was established in vivo, and histological evaluation and radiographic analysis were performed to verify the therapeutic effects of FOXA1 knockdown on bone healing. Results We identified 22 shared and differentially expressed miRNAs between hDPSC and hBMSC, 19 of which were downregulated in osteogenically induced samples. The miRNA-transcription factor interaction network showed that FOXA1 is the most significant and novel osteogenic differentiation biomarker among more than 300 transcription factors that is directly targeted by 12 miRNAs. FOXA1 knockdown significantly promoted hBMSC osteo-specific genes and increased mineral deposits in vitro. In addition, p-ERK1/2 levels were upregulated by FOXA1 silencing. Moreover, the increased osteogenic differentiation of FOXA1 knockdown hBMSCs was partially rescued by the addition of ERK1/2 signalling inhibitors. In a mouse model of femoral defects, a sheet of FOXA1-silencing BMSCs improved bone healing, as detected by microcomputed tomography and histological evaluation. Conclusion These findings collectively demonstrate that FOXA1 silencing promotes the osteogenic differentiation of BMSCs via the ERK1/2 signalling pathway, and silencing FOXA1 in vivo effectively promotes bone healing, suggesting that FOXA1 may be a novel target for bone healing.

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Stem Cell Research & Therapy

Tập 13 Số 1

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