Wnt signaling pathway correlates with ossification of the spinal ligament: A microRNA array and immunohistochemical...

Ossification of the posterior longitudinal ligament or the ligamentum flavum parallels endochondral ossification. Cell differentiation at the ossification front is known to be important during this process, although the factors regulating its initiation and progression are still unclear. The purpose of this study was to identify factors important for the regulation of chondrocyte/osteoblast differentiation during spinal ossification.

Ligamentum flavum tissue was isolated from 25 patients who underwent decompressive surgery for cervical ossification of the posterior longitudinal ligament. Tissue sections were used for in vitro culture to obtain primary cells through migration methods. To identify microRNAs associated with ossification of the posterior longitudinal ligament, cultured cells were prepared from the ligamentous tissue (n = 4; continuous type) or from control ligamentous samples harvested from patients with cervical spondylosis without spinal ossification, and analyzed using a microRNA array. The ligamentous sections were also examined by immunohistochemistry for the expression of candidate microRNA target genes.

The microRNA array identified 177 factors; 12 of which were expressed at significantly different levels in patients with ossification of the posterior longitudinal ligament compared to those in control patients. The hsa-miR-487b-3p was down-regulated in patients with ossification of the posterior longitudinal ligament, which met the false discovery rate of <0.05. This microRNA was predicted to regulate the expression of genes involved in Wnt signaling. Furthermore, immunohistochemistry of Wnt signaling proteins, including Wnt 3a, LRP5/6, and beta-catenin, revealed positive expression in mesenchymal cells and/or premature chondrocytes at the ossification front.

Our results suggested that down-regulation of miR-487b-3p plays an important role in the initiation of Wnt signaling during the ossification process. Wnt signaling may regulate both chondrocyte and osteoblast differentiation and the specification of endochondral ossification in the pathogenesis of ossification of the posterior longitudinal ligament or the ligamentum flavum.