Introduction: Intervertebral disc degeneration (IVDD) is a leading cause of Low back pain. Cellular senescence has been implicated in the pathogenesis of IVDD. Senescent nucleus pulposus (NP) cells accumulate in degenerative intervertebral disc, but further research is needed to understand the mechanisms underlying NP cell senescence. DNA G-quadruplexes (G4s) are non-canonical four-stranded nucleic acid structures formed by guanine-rich sequences, and are prevalent in the genome, particularly in telomeric regions and gene promoters where they play important roles in regulating gene expression. Despite their importance, the contribution of G4s in the progression of IVDD remains incompletely understood. This study aims to investigate NP cell senescence mechanisms and the role of G4s in intervertebral disc degeneration.
Methods: Western blotting, immunohistochemical staining, Enzyme-linked immunosorbent assay and Real-time quantitative polymerase chain reaction were used to detect KAZALD1 expression and senescence marker in human intervertebral disc specimens, natural aging mouse model, genomic G4 stabilized mouse model and human senescent NP cell. The regulatory effect of KAZALD1 on NP cell senescence was investigated using siRNA and recombinant human KAZALD1. SA-β-gal staining was used to evaluate NP cell senescence. Immunohistochemical fluorescence staining and immunohistochemical staining were used to detect genomic G4 folding. Fluorescence resonance energy transfer assay was used to confirm G4 formation of KAZALD1 potential G4 sequences. Dual-luciferase assay was conducted to assess the impact of G4 formation on KAZALD1 promoter activity.
Results: We revealed an interplay between G4 folding and NP cell senescence. Genomic G4 folding induced NP cell senescence, while senescent NP cells promoted further G4 folding. KAZALD1 was identified as a key participant in this interplay, with G4 formation in the KAZALD1 promoter leading to epigenetic suppression of its expression. Silencing KAZALD1 induced NP cell senescence, which could be reversed by reintroducing recombinant human KAZALD1. Furthermore, a significant decrease in KAZALD1 expression was observed in older individuals and in genomic G4 stabilized intervertebral discs and NP cells.
Discussion: The study demonstrates the involvement of KAZALD1 in the regulation of NP cell senescence through promoter G4 folding. These findings provide new insights into senescence-derived diseases such as IVDD.