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Long Non-coding RNA XIST May Influence Cervical Ossification of the Posterior Longitudinal Ligament Through Regulation of miR-17-5P/AHNAK/BMP2 Signaling Pathway

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Abstract

Long non-coding RNAs (lncRNAs) play an important role in the development of bone-related diseases. This study was conducted to investigate the role and mechanism of lncRNA X inactive specific transcript (XIST) in the occurrence of cervical ossification of the posterior longitudinal ligament (OPLL). Here, primary human ligament fibroblasts cells (LFCs) were isolated from 30 cases of OPLL and 30 normal cervical posterior longitudinal ligament (non-OPLL) tissues to perform the qPCR and Western blot assay. We found that the mRNA level of lncRNA XIST was significantly increased in OPLL LFCs compared to non-OPLL LFCs. By bioinformatics analysis, we found that lncRNA XIST has four binding sites for miR-17-5p and found that the mRNA level of miR-17-5p was also significantly decreased in OPLL LFCs compared to non-OPLL LFCs. Since AHNAK is the target gene of miR-17-5p, we further found that the expression of AHNAK was significantly reduced in non-OPLL LFCs after being transfected with miR-17-5p mimic. The qPCR results showed that the mRNA expressions of BMP2 and Runx2 were significantly decreased. After being transfected with lncRNA XIST siRNA in the non-OPLL LFCs, the mRNA levels of lncRNA XIST, AHNAK, BMP2, and Runx2 were significantly decreased and the phosphorylated protein of Smad1/5/8 was reduced. After being cultured by mechanical vibration, the mRNA levels of lncRNA XIST, AHNAK, BMP2, Runx2, COL1, OC, OPN, and Phospho1 were significantly increased, but the mRNA expression of miR-17-5p was significantly decreased. The expression of phosphorylated Smad1/5/8 protein was also significantly increased. Together, this study was the first to determine that XIST gene inhibition plays an important role in the occurrence of cervical OPLL, through the mechanism of regulation of miR-17-5P/AHNAK/BMP2 signaling pathway. Thus, XIST may be a potential target that could be modulated for the treatment of cervical OPLL.

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Abbreviations

OPLL:

Ossification of posterior longitudinal ligament

BMI:

Body mass index

lncRNAs:

Long non-coding RNAs

XIST:

X inactive specific transcript

miR-17-5p:

microRNA-17-5p

LFCs:

Ligament fibroblast cells

ceRNA:

Competing endogenous RNA

BMP2:

Bone morphogenic protein 2

IGF:

Insulin-like growth factor

bFGF:

Basic fibroblast growth factor

TGF-β:

Transforming growth factor-β

MAPK:

Mitogen-activated protein kinase

DANCR:

Differentiation antagonizing non-protein coding RNA

miR-141:

microRNA-141

miR-22:

microRNA-22

miR-200a:

microRNA-200a

EZH2:

Enhancer of Zeste homolog 2

RUNX2:

Runt-related transcription factor 2

miR-211:

microRNA-211

CXCR4:

C-X-C chemokine receptor type 4

COL I:

Collagen type I

OC:

Osteocalcin

OPN:

Osteopontin

Phospho1:

Phosphatase, orphan 1

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Acknowledgements

We would like to thank the patients and their caregivers for taking part in the study.

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Authors and Affiliations

  1. Spine Center, Department of Orthopaedics, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China

    Xinyuan Liao, Haisong Yang, Yu Chen, Deyu Chen, Lianshun Jia, Lili Yang & Xiongsheng Chen

  2. Institute of Spine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China

    Dezhi Tang

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  1. Xinyuan Liao
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  2. Dezhi Tang
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  4. Yu Chen
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  7. Lili Yang
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Contributions

Designed the study: LXY, TDZ, YLL, CXS. Performed the study: LXY, YHS, CY, CDY, JLS. Analyzed the data: LXY, TDZ, YLL, CXS. Wrote the paper: LXY, TDZ, YLL, CXS.

Corresponding authors

Correspondence to Dezhi Tang, Lili Yang or Xiongsheng Chen.

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Conflict of interest

Liao Xinyuan, Tang Dezhi, Yang Haisong, Chen Yu, Chen Deyu, Jia Lianshun, Yang Lili and Chen Xiongsheng declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This study was approved by the Ethics Committee of Shanghai Changzheng Hospital and conducted strictly in accordance with the Helsinki Declaration. All patients signed an informed consent for sample collection prior to study.

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Liao, X., Tang, D., Yang, H. et al. Long Non-coding RNA XIST May Influence Cervical Ossification of the Posterior Longitudinal Ligament Through Regulation of miR-17-5P/AHNAK/BMP2 Signaling Pathway. Calcif Tissue Int 105, 670–680 (2019). https://doi.org/10.1007/s00223-019-00608-y

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