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USF2 reduces BMP3 expression via transcriptional activation of miR-34a, thus promoting osteogenic differentiation of BMSCs

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Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

Osteoporosis is the most susceptible disease for people over 60. The main cause of osteoporosis is the decreased osteogenic differentiation of mesenchymal stem cells (MSCs). Here we showed that upstream stimulatory factor 2 (USF2)/microRNA-34a (miR-34a)/bone morphogenetic protein 3 (BMP3) axis regulated osteogenic differentiation of BMSCs.

Materials and methods

USF2 and miR-34a expression were examined using qPCR. Protein levels of BMP3 and osteogenic markers expression were evaluated using both western blot and qPCR. Activity of ALP was determined by ALP assay kit. Mineralization capacity of hBMSCs was assessed using ARS. Besides, CHIP assay was employed to verify whether USF2 could bind to miR-34a promoter. Finally, RIP assay and dual-luciferase reporter assay were employed to verify whether miR-34a directly bound to BMP3.

Results

Our results suggested that miR-34a was upregulated during osteogenic differentiation of BMSCs, and miR-34a overexpression could enhance osteogenic differentiation of BMSCs. USF2 could positively regulate miR-34a expression by interacting with its promoter. USF2 overexpression enhanced osteogenic differentiation of BMSCs, while miR-34a inhibition reversed the effect. Besides, BMP3 was the target of miR-34a. MiR-34a overexpression enhanced osteogenic differentiation of BMSCs, which was abolished by BMP3 overexpression.

Conclusion

Taken together, USF2 enhanced osteogenic differentiation of BMSCs via downregulating BMP3 by interacting with miR-34a promoter.

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Abbreviations

MSCs:

Mesenchymal stem cells

USF:

Upstream stimulatory factor

miR:

MicroRNA

BMP:

Bone morphogenetic protein

ALP:

Alkaline phosphatase

ARS:

Alizarin red staining

RIP:

RNA immunoprecipitation

qRT-PCR:

Quantitative real-time polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SD:

Standard deviation

ANOVA:

Analysis of variance

WB:

Western blot

OCN:

Osteocalcin

Osx:

Osterix

Runx2:

Runt-related transcription factor 2

IgG:

Immunoglobulin G

BMD:

Bone mineral density

BMC:

Bone mineral content

Tb.Th:

Thickness of trabeculae

Tb.N:

Number of trabeculae

Tb.Sp:

Separating degree of trabeculae

ELISA:

Enzyme-linked immunosorbent assay

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Acknowledgements

The authors express their gratitude to for his help for directing our article. This work was supported by Zhejiang Traditional Chinese Medicine Science and Technology Plan (2021ZA070), The Major Program of Zhejiang Traditional Chinese Medicine Science and Technology (2019ZZ012) and Zhejiang Traditional Chinese Medicine Modernization Special Project (2021ZX009).

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Authors and Affiliations

  1. Department of Orthopaedics, The Second Affiliated Hospital of Zhejiang Chinese Medical University, No.318, Chaowang Road, Gongshu District, Hangzhou, 310005, Zhejiang Province, China

    Han-Bing Zeng, Li-Qiang Dong, Yu-Liang Huang, Chao Xu, Xu-Hui Zhao & Lian-Guo Wu

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  1. Han-Bing Zeng
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  2. Li-Qiang Dong
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Contributions

HBZ: supervision, concepts, design, funding acquisition, preparation; LQD: experimental studies; YLH: experimental studies, data acquisition; CX: statistical analysis; XHZ: data analysis; LGW: editing, review; all the authors approved for the final version.

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Correspondence to Lian-Guo Wu.

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The authors declare that they have no conflict of interest.

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The protocol was permitted by the Animal Experimental Ethics Committee of The second affiliated hospital of Zhejiang Chinese medical university.

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Supplementary Information

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774_2021_1254_MOESM1_ESM.tif

Supplementary Figure S1. hBMSCs were transfected with oe-USF2 or pcDNA3.1. (A) Activity of ALP in hBMSCs was evaluated using ALP assay kit. (B) ARS of hBMSCs was observed and taken photos using a microscope, and ARS intensity was counted. (C) Runx2, Osx, Collagen I, OCN and ALP mRNA levels were assessed using qPCR. All the data represent mean ± SD of three replicate experiments. ** P < 0.01 or *** P < 0.001 (TIF 3478 KB)

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Zeng, HB., Dong, LQ., Huang, YL. et al. USF2 reduces BMP3 expression via transcriptional activation of miR-34a, thus promoting osteogenic differentiation of BMSCs. J Bone Miner Metab 39, 997–1008 (2021). https://doi.org/10.1007/s00774-021-01254-x

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  • DOI: https://doi.org/10.1007/s00774-021-01254-x

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