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LncRNA XIST facilitates S1P-mediated osteoclast differentiation via interacting with FUS

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Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

The diagnosis and treatment of osteoporosis, a frequent age-related metabolic bone disorder, remain incomprehensive and challenging. The potential regulatory role of lncRNA XIST and sphingosine kinase 1 (SPHK1) pathway need experimental investigations.

Materials and methods

RAW264.7 cells and BMMs were obtained for in vitro studies and 30 ng/mL RANKL was implemented for induction of osteoclast differentiation. The suppressing of lncRNA XIST, SPHK1 and fused in sarcoma (FUS) was achieved using small hairpin RNA, while overexpression of XIST and FUS was constructed by pcDNA3.1 vector system. Tartrate-resistant acid phosphatase (TRAP) staining was used for observation of formation of osteoclasts. RNA-pulldown analysis and RNA binding protein immunoprecipitation (RIP) was implemented for measuring mRNA and protein interactions. RT-qPCR was conducted to determining mRNA expression, whereas ELISA and Western blotting assay was performed for monitoring protein expression.

Results

RANKL induced osteoclast differentiation and upregulated expression of osteoclastogenesis-related genes that included NFATc1, CTSK, TRAP and SPHK1 and the level of lncRNA XIST in both RAW264.7 cells and BMMs. However, knockdown of lncRNA XIST or suppressing SPHK1 significantly reserved the effects of RANKL. LncRNA XIST was further demonstrated to be interacted with FUS and increased the stability of SPHK1, indicating its ability in promoting osteoclast differentiation through SPHK1/S1P/ERK signaling pathway.

Conclusion

LncRNA XIST promoted osteoclast differentiation via interacting with FUS and upregulating SPHK1/S1P/ERK pathway.

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Acknowledgements

This work was supported by Guangdong Natural Science Foundation Committee, Doctoral Startup Fund (2018a03010282).

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Authors and Affiliations

  1. Department of Spinal Surgery, The Fifth Affiliated Hospital of Sun Yat-Sen University, No. 52, Meihua East Road, Xiangzhou District, Zhuhai, 519000, Guangdong Province, China

    Da-Wei Zhang, Kui-Bo Zhang, Yuan-Qing Guo, Lian-Jun Yang & Hai Lv

  2. Department of Orthopaedic and Microsurgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510080, Guangdong Province, China

    Hong-Gang Wang

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  1. Da-Wei Zhang
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Contributions

(1) DWZ and LJY made substantial contributions to the conception. and design of the work; DWZ, HGW and KBZ are the acquisition, analysis, or interpretation of data; YQG is the creation of new software used in the work; (2) DWZ drafted the work or revised it critically for important intellectual content; (3) All authors approved the version to be published; (4) HL 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 Hai Lv.

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Supplementary Information

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774_2021_1294_MOESM1_ESM.tif

Supplementary file1 Suppressing SPHK1 had no effect on the lncRNA XIST expression in RAW264.7 cells and BMMs. A, B The expression of XIST measured by RT-qPCR and calculated via normalization to the level of GAPDH after knockdown of SPHK1 or treatment with SPHK1 inhibitor SKI-II. C The binding of lncRNA XIST and SPHK1 protein measured by RNA-pulldown assay. D The expression of XIST measured by RT-qPCR and calculated via normalization to the level of GAPDH after overexpression of SPHK1. ***P<0.001 (TIF 979 KB)

774_2021_1294_MOESM2_ESM.tif

Supplementary file2 LncRNA XIST reversed VPC23019 inhibiting osteoclast differentiation in RAW264.7 cells and BMMs. A The formation of osteoclasts detected by TRAP staining. B The level of S1P determined using ELISA assay. C The protein expression of SPHK1, S1PR1, S1PR3, p-ERK, ERK, NFATc1, CTSK and TRAP measured by Western blotting and calculated via normalization to the protein level of GAPDH. *P<0.05; **P<0.01; ***P<0.001 (TIF 2987 KB)

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Zhang, DW., Wang, HG., Zhang, KB. et al. LncRNA XIST facilitates S1P-mediated osteoclast differentiation via interacting with FUS. J Bone Miner Metab 40, 240–250 (2022). https://doi.org/10.1007/s00774-021-01294-3

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  • DOI: https://doi.org/10.1007/s00774-021-01294-3

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