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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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.
KeywordsOssification of the posterior longitudinal ligament (OPLL) lncRNA XIST miR-17-5p AHNAK BMP2 signaling
Ossification of posterior longitudinal ligament
Body mass index
Long non-coding RNAs
X inactive specific transcript
Ligament fibroblast cells
Competing endogenous RNA
Bone morphogenic protein 2
Insulin-like growth factor
Basic fibroblast growth factor
Transforming growth factor-β
Mitogen-activated protein kinase
Differentiation antagonizing non-protein coding RNA
Enhancer of Zeste homolog 2
Runt-related transcription factor 2
C-X-C chemokine receptor type 4
- COL I
Collagen type I
Phosphatase, orphan 1
We would like to thank the patients and their caregivers for taking part in the study.
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.
Compliance with Ethical Standards
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.
- 3.Li ZS, Zhang GB, Sheng HF et al (1999) Incidence of cervical ossification of the posterior longitudinal ligament in neck and shoulder pain patients in northern China. Chin J Spine Spinal Cord. 9(5):285–286 (in Chinese) Google Scholar
- 7.Kawaguchi Y, Furushima K, Sugimori K et al (2003) Association between polymorphism of the transforming growth factor-beta1 gene with the radiologic characteristic and ossification of the posterior longitudinal ligament. Spine 28(13):1424–1426Google Scholar
- 13.Nie M, Deng ZL, Liu J, Wang DZ (2015) Emerging roles for long noncoding RNAs in skeletal biology and disease. Biomed Res Int 2015:676575Google Scholar
- 15.Saydam O, Shen Y, Würdinger T, Senol O, Boke E, James MF, Tannous BA, Stemmer-Rachamimov AO, Yi M, Stephens RM, Fraefel C, Gusella JF, Krichevsky AM, Breakefield XO (2009) Downregulated microRNA-200a in meningiomas promotes tumor growth by reducing E-cadherin and activating the Wnt/beta-catenin signaling pathway. Mol Cell Biol 29(21):5923–5940CrossRefGoogle Scholar
- 28.Fang Y, Xu C, Fu Y (2015) MicroRNA-17-5p induces drug resistance and invasion of ovarian carcinoma cells by targeting PTEN signaling. J Biol Res 22(1):1–10Google Scholar
- 37.Tsukamoto N, Maeda T, Miura H et al (2006) Repetitive tensile stress to rat caudal vertebrae inducing cartilage formation in the spinal ligaments: a possible role of mechanical stress in the development of ossification of the spinal ligaments. Spine 5:234–242Google Scholar