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LINC02381, a sponge of miR-21, weakens osteogenic differentiation of hUC-MSCs through KLF12-mediated Wnt4 transcriptional repression

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Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

Human umbilical cord blood-derived MSCs (hUC-MSCs) have the potential to differentiate into osteoblasts. This study investigated the function and potential mechanisms of a novel lncRNA LINC02381 in hUC-MSC osteogenic differentiation.

Materials and methods

hUC-MSCs were maintained in osteogenic differentiation medium. RT-qPCR assay was performed to assess LINC02381 expression. Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining were performed to evaluate osteogenic differentiation. The interaction between miR-21 and LINC0238/KLF12 was determined by luciferase reporter and RNA immunoprecipitation (RIP) assays. Chromatin immunoprecipitation (ChIP) assay was used to confirm the transcriptional regulation of KLF12 on Wnt4 promoter. The nuclear translocation of β-catenin was evaluated using immunofluorescence. hUC-MSCs seeded on Bio-Oss Collagen scaffolds were transplanted into nude mice to assess in vivo osteogenesis. Bone formation was observed by H&E and Masson's trichrome staining. OSX and OPN levels were assessed by immunohistochemistry.

Results

LINC02381 was up-regulated in the clinical samples of osteoporotic patients. However, LINC02381 expression was reduced during osteogenic differentiation of hUC-MSCs. Enforced expression of LINC02381 suppressed the osteogenic differentiation of hUC-MSCs. Mechanistically, LINC02381 sponged miR-21 to enhance KLF12 expression, which led to the inactivation of Wnt/β-catenin signaling pathway. Furthermore, miR-21 mimics or KLF12 silencing counteracted LINC02381-induced inhibition of osteogenic differentiation, whereas IWP-4 (an inhibitor of Wnt pathway) abolished this effect.

Conclusion

In summary, LINC02381 repressed osteogenic differentiation of hUS-MSCs through sponging miR-21 to enhance KLF12-mediated inactivation of Wnt/β-catenin pathway, indicating that LINC02381 might be a therapeutic target for osteoporosis.

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Abbreviations

MSCs:

Mesenchymal stem cells

hUC-MSCs:

Human umbilical cord blood-derived MSCs

lncRNAs:

Long non-coding RNAs

sh-LINC02381:

ShRNA targeting LINC02381

KLF12:

Kruppel-like factor 12

sh-KLF12:

ShRNA targeting KLF12

sh-NC:

Negative control shRNA

ARS:

Alizarin red staining

ALP:

Alkaline phosphatase

RUNX2:

Runt-related transcription factor 2

OPN:

Osteopontin

OSX:

Osterix

WT:

Wild type

MUT:

Mutant

RIP:

RNA immunoprecipitation

ChIP:

Chromatin immunoprecipitation

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Acknowledgements

This work was supported by Yunnan Provincial Department of Science and Technology Kunming Medical University Applied Basic Research Joint Special Fund Project [2017FE468(-060)]

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Author notes

  1. Gang Zhao and Wen-Dong Luo are first co-authors.

Authors and Affiliations

  1. Department of Emergency Surgery, The Second Affiliated Hospital of Kunming Medical University, Yunnan Osteoporosis Research Center, Yunnan Trauma Surgery Research Center, Kunming, 650101, Yunnan, People’s Republic of China

    Gang Zhao, Wen-Dong Luo, Yong Yuan, Feng Lin, Li-Min Guo, Jing-Jing Ma, Han-Bo Chen, Huang Tang & Jun Shu

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Contributions

(1) JS made substantial contributions to the conception or design of the work; W-DL, HT and YY the acquisition, analysis, or interpretation of data; FL the creation of new software used in the work; (2) H-BC and HT drafted the work or revised it critically for important intellectual content; (3) L-MG and J-JM approved the version to be published; (4) GZ agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Correspondence to Jun Shu.

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The experimental procedures were approved by the Ethics Committee of the Second Affiliated Hospital of Kunming Medical University. All subjects provided written informed consents. All animal experiments were approved by the Animal Ethical Committee of the Second Affiliated Hospital of Kunming Medical University.

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774_2021_1277_MOESM1_ESM.tif

(A) The effects of LINC02381 overexpression on theTCF/LEF reporter activity. (B) The KLF12 binding sites in the Wnt4 promoter. **p <0.01. (TIF 1731 kb)

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Zhao, G., Luo, WD., Yuan, Y. et al. LINC02381, a sponge of miR-21, weakens osteogenic differentiation of hUC-MSCs through KLF12-mediated Wnt4 transcriptional repression. J Bone Miner Metab 40, 66–80 (2022). https://doi.org/10.1007/s00774-021-01277-4

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  • DOI: https://doi.org/10.1007/s00774-021-01277-4

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