MicroRNA-181 regulates the development of Ossification of Posterior longitudinal ligament via Epigenetic Modulation by targeting PBX1

• Published in: Theranostics 2020-01, Vol.10 (17), p.7492-7509
• Main Authors: Liu, Ning, Zhang, Zicheng, Li, Li, Shen, Xiaolong, Sun, Baifeng, Wang, Ruizhe, Zhong, Huajian, Shi, Qianghui, Wei, Leixin, Zhang, Yizhi, Wang, Yue, Xu, Chen, Liu, Yang, Yuan, Wen
• Format: Article
• Language: English
• Subjects: Acetylation - drug effects; Adult; Aged; Animals; Antagomirs - administration & dosage; Cell culture; Cells, Cultured; Cloning; Computational Biology; Disease; Disease Models, Animal; DNA Methylation - drug effects; Epigenesis, Genetic - drug effects; Epigenetics; Female; Gene expression; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Histones - genetics; Humans; Ligaments; Longitudinal Ligaments - cytology; Longitudinal Ligaments - diagnostic imaging; Longitudinal Ligaments - pathology; Longitudinal Ligaments - surgery; Male; Mice; MicroRNAs; MicroRNAs - agonists; MicroRNAs - antagonists & inhibitors; MicroRNAs - metabolism; Middle Aged; Ossification of Posterior Longitudinal Ligament - genetics; Ossification of Posterior Longitudinal Ligament - pathology; Ossification of Posterior Longitudinal Ligament - surgery; Patients; Pre-B-Cell Leukemia Transcription Factor 1 - genetics; Primary Cell Culture; Research Paper; X-Ray Microtomography
• ISSN: 1838-7640; 1838-7640
• DOI: 10.7150/thno.44309

Ossification of the posterior longitudinal ligament (OPLL) presents as the development of heterotopic ossification in the posterior longitudinal ligament of the spine. The etiology of OPLL is genetically linked, as shown by its high prevalence in Asian populations. However, the molecular mechanism of the disease remains obscure. In this study, we explored the function and mechanism of OPLL-specific microRNAs. The expression levels of the ossification-related OPLL-specific miR-181 family were measured in normal or OPLL ligament tissues. The effect of miR-181a on the ossification of normal or pathogenic ligament cells was tested using real-time polymerase chain reaction (PCR), Western blot, alizarin red staining and alkaline phosphatase (ALP) staining. The candidate targets of miR-181 were screened using a dual luciferase reporter assay and functional analysis. The link between miR-181a and its target PBX1 was investigated using chromatin immunoprecipitation, followed by real-time PCR detection. Histological and immunohistochemical analysis as well as micro-CT scanning were used to evaluate the effects of miR-181 and its antagonist using both tip-toe-walking OPLL mice and bone formation assays. Using bioinformatic analysis, we found that miR-181a-5p is predicted to play important roles in the development of OPLL. Overexpression of miR-181a-5p significantly increased the expression of ossification-related genes, staining level of alizarin red and ALP activity, while the inhibition of miR-181a-5p by treatment with an antagomir had the opposite effects. Functional analysis identified PBX1 as a direct target of miR-181a-5p, and we determined that PBX1 was responsible for miR-181a-5p's osteogenic phenotype. By chromatin immunoprecipitation assay, we found that miR-181a-5p controls ligament cell ossification by regulating PBX1-mediated modulation of histone methylation and acetylation levels in the promoter region of osteogenesis-related genes. Additionally, using an model, we confirmed that miR-181a-5p can substantially increase the bone formation ability of posterior ligament cells and cause increased osteophyte formation in the cervical spine of tip-toe-walking mice. Our data unveiled the mechanism by which the miR-181a-5p/PBX1 axis functions in the development of OPLL, and it revealed the therapeutic effects of the miR-181a-5p antagomir in preventing OPLL development both and . Our work is the first to demonstrate that microRNA perturbation could modulate the