IGF-2 Modified by the m6A Demethylation Enzyme ALKBH5 in the Ossification of the Ligamentum Flavum

Abstract

Background: Ossification of the ligamentum flavum (OLF) is a pathological heterotopic ossification of the paravertebral ligament. However, the specific pathophysiology mechanism of this disease is still unknown. The m6A methylation and its potential functions in OLF remain to be unexplored. Method: In this study, we performed a transcriptome-wide methylation analysis using the OLF and normal ligaments to explore the mechanism of OLF. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. We screened out the methylase-ALKBH5, and by promoting or inhibiting its expression, observing the content of m6A, and measuring the ossification of ligaments, and then measuring IGF expression situation by alizarin red staining, alkaline phosphatase, immunofluorescence, immunohistochemistry, etc. Result: MeRIP-seq and qPCR showed that the m6A methylation level of OLF group was usually higher than that of control group. In addition, we found that ALKBH5 is an important demethyltransferase in OLF, which promotes the expression of m6A. ALKBH5 promotes the expression of IGF-2, which in turn promotes osteogenesis in OLF.Conclusion: Overall, we provided a region-specific map of m6A methylation and characterized the distinct features of specific and common methylation in OLF, and we proved that IGF-2 can be regulated by ALKBH5 to promote the process of OLF.