Identification of Potential Targets for EGFR-Regulated Nucleus Pulposus Degeneration Using Single-Cell RNA Sequencing and Machine Learning.

Abstract

Introduction: While nucleus pulposus cell (NPC) degeneration is a primary driver of intervertebral disc degeneration (IVDD), the cellular heterogeneity and molecular interactions underlying NPC degeneration remain poorly characterized. Previous studies have shown that EGFR signaling plays a significant role in NPC differentiation and collagen matrix production. Consequently, this study aims to identify the critical downstream regulatory molecule of EGFR in the process of NPC degeneration.

Methods: We conducted subpopulation identification and functional analysis on scRNA-seq results in the GSE165722 dataset. Through pseudotime analysis, we identified genes with significant changes. Furthermore, we performed single-gene GSEA based on EGFR expression levels and conducted WGCNA to identify hub genes. Then, a combination of three machine learning algorithms (Lasso, XGBoost, and Random Forest), ROC curve, and validation in clinical specimens was employed to identify potential downstream regulatory molecule of EGFR associated with NPC degeneration. Finally, the regulatory effect of EGFR on potential downstream molecules was validated through both in vitro and in vivo experiments.

Results: NPC from six severe IVDD samples were classified into six subpopulations, among which Fib-NPC was identified by functional and pseudotime analyses as a late-stage degenerative subpopulation linked to IVDD, with upregulated EGFR expression observed during degeneration. Five hub genes were identified through the intersection of pseudotime analysis, single-gene GSEA, and WGCNA. By integrating the results from three machine learning and validating through ROC curve and IHC, JAK1 was further identified as a downstream regulatory target of EGFR. Then, we found that JAK1 expression was elevated in NPC under oxidative stress, but remained unchanged following Gefitinib pretreatment. We further developed an IVDD model using mice with NP-specific inactivation of EGFR, which demonstrated that EGFR inactivation attenuated the upregulation of JAK1 in the degenerated NP region.

Conclusion: This study reveals a significant degenerative process in NPC and indicates that EGFR may contribute to this degeneration by regulating JAK1, thereby identifying a potential therapeutic target for delaying IVDD.