Identification and Validation of Circadian Rhythm-Related Genes Involved in Intervertebral Disc Degeneration and Analysis of Immune Cell Infiltration via Machine Learning

IF 3.4 3区医学 Q1 ORTHOPEDICS

JOR Spine Pub Date : 2025-04-13 DOI:10.1002/jsp2.70066

Yongbo Zhang, Liuyang Chen, Sheng Yang, Rui Dai, Hua Sun, Liang Zhang

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Abstract

Background

Low back pain is a significant burden worldwide, and intervertebral disc degeneration (IVDD) is identified as the primary cause. Recent research has emphasized the significant role of circadian rhythms (CRs) and immunity in affecting intervertebral discs (IVD). However, the influence of circadian rhythms and immunity on the mechanism of IVDD remains unclear. This study aimed to identify and validate key rhythm-related genes in IVDD and analyze their correlation with immune cell infiltration.

Methods

Two gene expression profiles related to IVDD and rhythm-related genes were obtained from the Gene Expression Omnibus and GeneCards databases to identify differentially expressed rhythm-related genes (DERGs). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) were conducted to explore the biological functions of these genes. LASSO regression and SVM algorithms were employed to identify hub genes. We subsequently investigated the correlation between hub rhythm-related genes and immune cell infiltration. Finally, nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated from normal and degenerative human IVD tissues. Hub rhythm-related genes expression in NPMSCs was confirmed by real-time quantitative PCR (RT-qPCR).

Results

Six hub genes related to CRs (CCND1, FOXO1, FRMD8, NTRK2, PRRT1, and TFPI) were screened out. Immune infiltration analysis revealed that the IVDD group had significantly more M0 macrophages and significantly fewer follicular helper T cells than those of the control group. Specifically, M0 macrophages were significantly associated with FRMD8, PRRT1, and TFPI. T follicular helper cells were significantly associated with FRDM8, FOXO1, and CCND1. We further confirmed that CCND1, FRMD8, NTRK2, and TFPI were dysrhythmic within NPMSCs from degenerated IVD in vitro.

Conclusion

Six genes (CCND1, FOXO1, FRMD8, NTRK2, PRRT1 and TFPI) linked to circadian rhythms associated with IVDD progression, together with immunity. The identification of these DEGs may provide new insights for the diagnosis and treatment of IVDD.

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通过机器学习识别和验证与椎间盘退变有关的昼夜节律相关基因和免疫细胞浸润分析

背景：腰痛是世界范围内的一个重要负担，椎间盘退变（IVDD）被确定为主要原因。最近的研究强调了昼夜节律（CRs）和免疫在影响椎间盘（IVD）中的重要作用。然而，昼夜节律和免疫对IVDD机制的影响尚不清楚。本研究旨在鉴定和验证IVDD中关键的节律相关基因，并分析其与免疫细胞浸润的相关性。方法从基因表达Omnibus和GeneCards数据库中获取IVDD和节律相关基因的两个基因表达谱，鉴定差异表达节律相关基因（derg）。通过基因本体（GO）、京都基因与基因组百科全书（KEGG）和基因集富集分析（GSEA）对这些基因的生物学功能进行了探讨。采用LASSO回归和SVM算法对轮毂基因进行识别。我们随后研究了中枢节律相关基因与免疫细胞浸润之间的相关性。最后，从正常和退行性人IVD组织中分离出髓核源间充质干细胞（NPMSCs）。实时定量PCR （RT-qPCR）证实了中枢节律相关基因在NPMSCs中的表达。结果共筛选到6个与CRs相关的中枢基因（CCND1、FOXO1、FRMD8、NTRK2、PRRT1和TFPI）。免疫浸润分析显示，IVDD组M0巨噬细胞明显多于对照组，滤泡辅助性T细胞明显少于对照组。具体来说，M0巨噬细胞与FRMD8、PRRT1和TFPI显著相关。T滤泡辅助细胞与FRDM8、fox01和CCND1显著相关。我们进一步证实，CCND1、FRMD8、NTRK2和TFPI在体外退行性IVD的NPMSCs中存在节律异常。结论6个基因（CCND1、FOXO1、FRMD8、NTRK2、PRRT1和TFPI）与IVDD进展相关的昼夜节律以及免疫相关。这些deg的鉴定可能为IVDD的诊断和治疗提供新的见解。

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