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lncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/β-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification

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Abstract

Background

Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms.

Results

On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/β-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/β-catenin axis.

Conclusions

Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

HO:

Heterotopic ossification

TDSCs:

Tendon stem cells

BMSCs:

Bone marrow mesenchymal cells

MEG3:

Maternally expressed gene 3

TCF4:

Transcription factor 4

FBS:

Fetal Bovine Serum

PDGFRα:

Platelet-derived growth factor receptor-α

EDTA:

Ethylenediaminetetraacetic acid

FISH :

Fluorescence in situ hybridization

IHC:

Immunohistochemistry

micro-CT:

Micro-computed tomography

ECL:

Electrochemiluminescence

BCA:

Bicinchoninic acid

TBST:

Tris buffered saline tween

AAV9:

Adeno-associated virus 9

ALP:

Alkaline phosphatase

ARS:

Alizarin Red S

RIP:

RNA Immunoprecipitation

HE:

Hematoxylin and eosin

PBS:

Phosphate-buffered saline

qRT-PCR:

Quantitative reserve-transcriptase polymerase chain reaction

SD:

Standard deviation

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Acknowledgements

We appreciated the support from Base for Interdisciplinary Innovative Talent Training, Shanghai Jiao Tong University and Youth Science and Technology Innovation Studio of Shanghai Jiao Tong University School of Medicine.

Funding

This study were supported by National Natural Science Foundation of China (81830076, 8217090787); Shanghai Engineering Technology Research Center and Professional Technology Service Platform project of 2020 “Science and Technology Innovation Action Plan” of Shanghai (20DZ2254100); Municipal Hospital Clinical Skills and Innovation Capacity of Three-year Action Plan Program of Shanghai Shenkang Hospital Development Center (SHDC2020CR2039B, SHDC2020CR6019-002); Biomedical Technology Support Special Project of Shanghai “Science and Technology Innovation Action Plan” (20S31900300, 21S31902300); Clinical Research Center (CRC) of Shanghai University of Medicine and Health Sciences (20MC2020001); Research on transformation chain and transformation mode of scientific and technological achievements in public hospitals-Take Class A tertiary hospital in Shanghai as an example (lygl202214).

All the funding body have not participated in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Authors and Affiliations

  1. Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, People’s Republic of China

    Hang Liu, Ziyang Sun, Gang Luo, Hongjiang Ruan, Bing Tu, Juehong Li & Cunyi Fan

  2. Shanghai Engineering Research Center for Orthopaedic Material Innovation and Tissue Regeneration, Shanghai, 201306, People’s Republic of China

    Hang Liu, Ziyang Sun, Gang Luo, Hongjiang Ruan, Bing Tu, Juehong Li & Cunyi Fan

  3. Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, People’s Republic of China

    Yuehao Hu

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Contributions

HL and ZYS designed and conducted the in vitro and in vivo experiments, analyzed the data and wrote the manuscript.

YHH conducted some of the in vivo experiments, micro-CT, IHC, IF and WB analysis.

GL, XW and ZJT conducted some of the in vitro experiments, ALP, ARS, IF and WB analysis.

HJR conducted some of experiments of PCR and ELISA.

BT and ZYS conducted some of experiments of animal model and cell culture.

CYF designed and conducted the research, wrote the manuscript, directed the project, and provided funding.

JHL conducted the research, wrote the manuscript.

HL, ZYS, JHL and CYF have read and verified the underlying data.

All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Juehong Li or Cunyi Fan.

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All experiments and procedures were approved by the ethics committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.

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Gang Luo and Yuehao Hu contribute equally to this work as co-first authors.

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Liu, H., Sun, Z., Luo, G. et al. lncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/β-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification. Stem Cell Rev and Rep 19, 2311–2328 (2023). https://doi.org/10.1007/s12015-023-10562-w

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