In the top panel of Fig. 4D, the bottom part of Stage I and the top part of Stage II-III images show an overlap. This is permissible since spermatogenesis is a continuous process from stage I to stage XII in seminiferous tubules and the panels are all from the same animal. The authors have, however, added an explanation to the legend to avoid any misunderstanding. The authors mistakenly used the wrong merge image for WT histone H4 in the bottom row when assembling Fig. 2C. The corrected and original figure panels are shown below.
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.
Ossification of the posterior longitudinal ligament (OPLL) is an emerging spinal disease caused by heterotopic ossification of the posterior longitudinal ligament. The pathological mechanism is poorly understood, which hinders the development of nonsurgical treatments. Here, we set out to explore the function and mechanism of small extracellular vesicles (sEVs) in OPLL. Global miRNA sequencings are performed on sEVs derived from ligament cells of normal and OPLL patients, and we have showed that miR-320e is abundantly expressed in OPLL-derived sEVs compare to other sEVs. Treatment with either sEVs or miR-320e significantly promote the osteoblastic differentiation of normal longitudinal ligament cells and mesenchymal stem cells and inhibit the osteoclastic differentiation of monocytes. Through a mechanistic study, we find that TAK1 is a downstream target of miR-320e, and we further validate these findings in vivo using OPLL model mice. Together, our data demonstrate that OPLL ligament cells secrete ossification-promoting sEVs that contribute to the development of ossification through the miR-320e/TAK1 axis.
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