Abstract
Ossification of the posterior longitudinal ligament (OPLL) presents as pathological heterotopic ossification of the spinal ligaments. However, its underlying molecular mechanism is still unclear. Our previous findings suggested that altered microRNA regulatory network are critical for the development of OPLL. Here, we set out to unveiling the detailed mechanism of those altered OPLL-specific microRNAs. We screened a set of differentially expressed OPLL-specific microRNAs from the previous sequencing data and showed that microRNA-10a actively modulates the ossification of posterior ligament cells in vitro. Using a tissue-engineered scaffold grown from 4-week-old BALB/c homozygous nude mice, we found that altered microRNA-10a expression in posterior ligament cells indeed affected the heterotopic bone formation in vivo. Furthermore, computational analysis showed that the negative ossification regulator ID3 is a functional target gene of microRNA-10a, and its expression was also significantly altered during microRNA-10a modulation both in vitro and in vivo. Also, we have demonstrated that the ossification promoting function of microRNA-10a requires ID3, as ID3 actively inhibits RUNX2. Thus, we identified a critical role for highly altered OPLL-specific microRNA-10a in regulating the development of OPLL by modulating the ID3/RUNX2 axis.
Highlights
Ossification of the posterior longitudinal ligament (OPLL) is a common spinal disease that often causes severe injuries and compressions to the spinal cord and nerve roots
We validated the expression changes of these miRNAs using real-time polymerase chain reaction (PCR) analysis of OPLL (n = 11; twelve samples were used, but one failed to extract high quality RNAs) and PLL (n = 9) tissue samples and showed that the results were consistent with the generation sequencing (NGS) data (Fig. 1B)
We found that the expression changes of these two negative regulators of ossification were inversely correlated with the ossification-related factors RUNX2, alkaline phosphatase (ALP) and OCN between OPLL and PLL cells (Fig. 4B), while miR-10a-3p was inversely correlated with the candidates (Figs 4C and S1A–C)
Summary
Ossification of the posterior longitudinal ligament (OPLL) is a common spinal disease that often causes severe injuries and compressions to the spinal cord and nerve roots. MiR-690 activated by RUNX2 positively regulates RUNX2-induced osteogenic differentiation by inactivating the NF-κB pathway via the downregulation of subunit p657, whereas TNF-α-induced NF-kB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin[8] These results suggest that osteogenesis is tightly controlled by miRNAs, and miRNA regulation may be an upstream mediator of the osteogenic process. A recent study by our group defined OPLL-specific microRNAs (miRNAs) using high-throughput miRNA sequencing and revealed that their predicted regulatory network was closely related to ossification development[9]. Their specific roles in OPLL development needs further identification. Our work here demonstrated the important role of miR-10a-3p in the ossification process of OPLL, and revealed its mechanism through the ID2/RUNX2 axis, which may further contributes to the understanding of OPLL development
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