Dataset Information

The microRNA-10a/ID3/RUNX2 axis modulates the development of Ossification of Posterior Longitudinal Ligament.

ABSTRACT: Ossification of the posterior longitudinal ligament (OPLL) presents as pathological heterotopic ossification of the spinal ligaments. However, its underlying molecular mechanism is still unclear. Our previous findings suggested that altered microRNA regulatory network are critical for the development of OPLL. Here, we set out to unveiling the detailed mechanism of those altered OPLL-specific microRNAs. We screened a set of differentially expressed OPLL-specific microRNAs from the previous sequencing data and showed that microRNA-10a actively modulates the ossification of posterior ligament cells in vitro. Using a tissue-engineered scaffold grown from 4-week-old BALB/c homozygous nude mice, we found that altered microRNA-10a expression in posterior ligament cells indeed affected the heterotopic bone formation in vivo. Furthermore, computational analysis showed that the negative ossification regulator ID3 is a functional target gene of microRNA-10a, and its expression was also significantly altered during microRNA-10a modulation both in vitro and in vivo. Also, we have demonstrated that the ossification promoting function of microRNA-10a requires ID3, as ID3 actively inhibits RUNX2. Thus, we identified a critical role for highly altered OPLL-specific microRNA-10a in regulating the development of OPLL by modulating the ID3/RUNX2 axis.

SUBMITTER: Xu C

PROVIDER: S-EPMC6003989 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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The microRNA-10a/ID3/RUNX2 axis modulates the development of Ossification of Posterior Longitudinal Ligament.

Xu Chen C Zhang Hao H Gu Wei W Wu Huiqiao H Chen Yuanyuan Y Zhou Wenchao W Sun Baifeng B Shen Xiaolong X Zhang Zicheng Z Wang Yue Y Liu Yang Y Yuan Wen W

Scientific reports 20180615 1

Ossification of the posterior longitudinal ligament (OPLL) presents as pathological heterotopic ossification of the spinal ligaments. However, its underlying molecular mechanism is still unclear. Our previous findings suggested that altered microRNA regulatory network are critical for the development of OPLL. Here, we set out to unveiling the detailed mechanism of those altered OPLL-specific microRNAs. We screened a set of differentially expressed OPLL-specific microRNAs from the previous sequen ...[more]

PMID: 29907859

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Project description:Objectives: 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. Methods: 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 in vivo bone formation assays. Results: 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 in vivo 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. Conclusions: 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 in vivo and in vitro. Our work is the first to demonstrate that microRNA perturbation could modulate the development of OPLL through epigenetic regulation.

Project description:Ossification of the posterior longitudinal ligament (OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 (CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7 levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin-proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.

Dataset Information

The microRNA-10a/ID3/RUNX2 axis modulates the development of Ossification of Posterior Longitudinal Ligament.

Publications

The microRNA-10a/ID3/RUNX2 axis modulates the development of Ossification of Posterior Longitudinal Ligament.

Similar Datasets

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