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

Abstract

Background: The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remains 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.