Abstract
BackgroundThe 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.MethodsExpression 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.ResultsIn 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.ConclusionsBeclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.
Highlights
Ossification of the posterior longitudinal ligament (OPLL) is characterized by heterotopic ossification in the spine ligament [1, 2]
Autophagy of osteocytes was first confirmed by Zahm et al [10], and subsequent studies had reported that autophagic receptor and Unc-51 like autophagy activating kinase 1 (ULK1) had an important effect on the activity and differentiation of osteoblasts [11,12,13]
Correlation between rate of autophagy and osteogenic differentiation markers correlation was analyzed between the expression of autophagy-related genes (LC3 and Beclin1 mRNA) and osteogenic differentiation-related genes
Summary
Ossification of the posterior longitudinal ligament (OPLL) is characterized by heterotopic ossification in the spine ligament [1, 2]. A macromolecular degradation process, plays a pivotal role in the maintenance of cell differentiation [9]. Another study demonstrated that osteoblastic differentiation was dependent on Beclin dependent autophagy [14]. It is generally accepted that autophagy plays an essential role in the osteogenic differentiation of mesenchymal stem cells [14, 18]. Autophagy of spinal ligament fibroblast has not yet been investigated and the underlying role of autophagy in OPLL processes remains unknown. 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
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