Molecular Pathophysiology of Ossification of the Posterior Longitudinal Ligament (OPLL)

Nam, Dae Cheol; Lee, Hyun Jae; Lee, Choong Jae; Hwang, Sun‐Chul

doi:10.4062/biomolther.2019.043

Cited by 48 publications

(43 citation statements)

References 86 publications

(97 reference statements)

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“…OPLL is a common spinal disease that can lead to symptoms of spinal cord compression and radiculopathy as the result of a mass of ectopic ossification developing in the posterior longitudinal ligament of the spine 16 . Since no effective measures have been developed to inhibit or reverse the progression of the disease, it is urgent to find critical disease regulatory targets for developing gene therapies 17 . In this study, by using in vitro experiments and in vivo OPLL models, we uncovered the function and mechanism of OPLL-related miR-181a-5p.…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Zhang

et al. 2020

Theranostics

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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.

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Section: Discussionmentioning

confidence: 99%

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

Liu

Zhang

et al. 2020

Theranostics

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“…Ossi cation of the posterior longitudinal ligament (OPLL), de ned as an ectopic ossi cation (calci cation), would lead to paralysis of extremities and disturbances of motility [18]. Up to now, the osteo-lineage derivation, cell atlas transformation and molecular regulation have never been elucidated, which retarded the development of etiological treatment [18][19][20]. Single-cell platform, taking advantage of horizontal cell atlas and vertical trajectory analysis, would broaden the horizons in this eld.…”

Section: Discussionmentioning

confidence: 99%

Systematic Study of Single-Cell Isolation from Musculoskeletal Tissues for Single-Cell Sequencing

Guo¹,

Liu²,

Zhang³

et al. 2020

Preprint

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Background: The single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus, tendons, etc. as well as their pathological state, which are tense and dense, it’s full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed specific enzymatic treatment protocols for several human pathological musculoskeletal tissues, including degenerated nucleus pulposus, ossifying posterior longitudinal ligament and knee articular cartilage with osteoarthritis, aiming to rapidly and efficiently harvest qualified single-cell suspensions to meet the requirements of single-cell RNA-sequencing (scRNA-seq).Results: The single-cell suspensions from human degenerated nucleus pulposus and ossifying posterior longitudinal ligaments were both qualified after systematic quality control. Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library. A clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. However, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets. Conclusions: In conclusion, we provided rapid and efficient single-cell isolation protocols for human degenerated nucleus pulposus and ossifying posterior longitudinal ligament, which could be applied for scRNA-seq. More efforts will be made on improving the protocols for human articular cartilage.

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“…To confirm this hypothesis, a clinical study was carried out on 37 subjects (21 cases of OPLL patients and 16 cases of non-OPLL patients). The main objectives of this research are as follows: (1) to compare the autophagy level of spinal ligament fibroblasts between OPLL and non-OPLL patients and (2) to investigate the association between autophagy level and the expression of osteogenic differentiation genes.…”

Section: Introductionmentioning

confidence: 99%

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

et al. 2020

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Background The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods Expression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001), and collagen type 1 (COL 1) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% (P < 0.01), 52% (P < 0.01), and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

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Molecular Pathophysiology of Ossification of the Posterior Longitudinal Ligament (OPLL)

Cited by 48 publications

References 86 publications

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

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

Systematic Study of Single-Cell Isolation from Musculoskeletal Tissues for Single-Cell Sequencing

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

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