Analysis of EZH2 target genes in senescent nucleus pulposus mesenchymal stem cells by integrating CUT&Tag and RNA-seq data [RNA-seq]
Intervertebral disc degeneration (IVDD) is one of the leading factors for low back pain, which has an enormous socioeconomic burden. Nucleus pulposus (NP) tissue is the core unit of the intervertebral disc (IVD) and contains nucleus pulposus mesenchymal stem cells (NPMSCs). NPMSCs are important for the health of NP tissue and IVDs. In the process of IVDD, NPMSCs undergo cellular senescence under the influence of various unfavourable factors, but the specific molecular mechanism remains unclear. Enhancer of Zeste Homologue 2 (EZH2) is a transcriptional regulator associated with cell senescence and can bind to the promoter region of downstream genes or transcription factors. In this study, decreased EZH2 expression in senescent NPMSCs from both human and rat IVDD specimens was observed, human nucleus pulposus mesenchymal stem cells (hNPMSCs) were isolated from human NP tissues, and tert-butyl hydroperoxide (TBHP) was used to induce hNPMSC senescence. The expression levels of P16 and P21 were significantly increased in senescent hNPMSCs, and the expression of EZH2 was significantly reduced. To elucidate the function of EZH2 in IVDD, RNA sequencing (RNA-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) were used to analyse the gene expression changes and the target genes of EZH2 in senescent hNPMSCs. The cross analysis of RNA-seq and CUT&Tag results showed that 58 target genes were directly regulated by EZH2, including 16 upregulated genes and 42 downregulated genes. Finally, protein-protein interaction (PPI) network analysis identified the hub genes associated with the mitotic cell cycle and cell proliferation, including threonine tyrosine kinase (TTK), minichromosome maintenance complex component 6 (MCM6), Aurora Kinase B (AURKB), Non-SMC condensin I complex subunit G (NCAPG), and cell division cycle associated 8 (CDCA8). In summary, this study analysed the genomic targets and hub gene network of EZH2 in senescent NPMSCs. The hub gene network revealed the important role of EZH2 in mitosis and cell cycle process. The decreased expression of EZH2 and hub genes might be one of the causes of hNPMSC senescence. Targeting EZH2 may be a potential treatment for IVDD, but the transcriptional regulatory function of EZH2 in IVDD still needs to be investigated further. Overall design: To study the changes in gene expression levels in senescent hNPMSCs, RNA-seq was performed on the cells of the senescent hNPMSC group (75 µM treatment) and control group.