Identification and immune characteristics of N6-methyladenosine related ferroptosis-related genes in intervertebral disc degeneration.

The damage of ferroptosis is related to the pathogenesis of intervertebral disc degeneration (IDD). N6-methyladenosine (m6A) modification accounts for more than 80% of RNA modifications in eukaryotic cells. However, the key role of m6A related ferroptosis-related genes (FRGs) in IDD remain explorable. Firstly, batch correction between datasets was performed. Weighted gene co-expression network analysis (WGCNA) was then conducted to acquire the most relevant module genes. Differentially expressed genes (DEGs) between the IDD and Normal Group were obtained by differential analysis subsequently. Next, according to the Pearson correlation and the intersection of m6A and FRGs related genes, m6A-FRGs related genes were selected. Additionally, we intersected module genes, DEGs and m6A-FRGs related genes to obtain DEGs of m6A-FRGs related module genes (m6A-FRGs-DEGs). Least absolute shrinkage and selection operator (LASSO) was applied to get HUBgenes. After, we conducted gene set enrichment analysis (GSEA) to gain function items and related pathways of HUBgenes. Single sample gene set enrichment analysis (ssGSEA) and wilcox.test were proceeded to analyse differences in relative abundance of immune cells between the IDD and Normal Groups, and a nomogram was constructed based on significantly different HUBgenes. The Comparative Toxicogenomics Database (CTDbase) was then applied to predict potential drugs or molecular compounds that could modulate HUBgenes. Last but not least, we performed the quantitative real-time fluorescence PCR (qRT-PCR) to verify HUBgenes. 181 m6A-FRGs-DEGs were acquired by the intersection of key module genes (from MEtan, MEsalmon, MEbrown and MEgreen), 362 DEGs and 15,678 m6A-FRGs related genes. Subsequent analysis showed that HUBgenes (ZNF595, PLXDC1, FNBP1L, KLRB1, NRCAM, PPCDC, C9orf139, SIGLEC17P, RRAS2 and DPRXP4) significantly participated in positive regulation of cytokine production, mitochondrial inner membrane and organellar ribosome. Besides, the relative abundance of neutrophils was found significantly different between IDD and normal groups. A nomogram was constructed based on ZNF595 and RRAS2, and there were 11 drugs targeted on ZNF595, while 118 drugs predicted based on RRAS2 such as Tetrachlorodibenzodioxin, Bisphenol A and Benzo(a)pyrene. Lastly, ZNF595 and RRAS2 were both obviously up-regulated in IDD according to the qRT-PCR. Our research suggested 10 HUBgenes were significantly associated with IDD, providing more evidence about the vital role of HUBgenes in IDD.