Identification and validation of ferroptosis-related biomarkers in intervertebral disc degeneration.

Introduction: Ferroptosis plays a significant role in intervertebral disc degeneration (IDD). Understanding the key genes regulating ferroptosis in IDD could reveal fundamental mechanisms of the disease, potentially leading to new diagnostic and therapeutic targets. Methods: Public datasets (GSE23130 and GSE70362) and the FerrDb database were analyzed to identify ferroptosis-related genes (DE-FRGs) involved in IDD. Single-cell RNA sequencing data (GSE199866) was used to validate the specific roles and expression patterns of these genes. Immunohistochemistry and Western blot analyses were subsequently conducted in both clinical samples and mouse models to assess protein expression levels across different tissues. Results: The analysis identified seven DE-FRGs, including MT1G, CA9, AKR1C1, AKR1C2, DUSP1, CIRBP, and KLHL24, with their expression patterns confirmed by single-cell RNA sequencing. Immunohistochemistry and Western blot analysis further revealed that MT1G, CA9, AKR1C1, AKR1C2, DUSP1, and KLHL24 exhibited differential expression during the progression of IDD. Additionally, the study highlighted the potential immune-modulatory functions of these genes within the IDD microenvironment. Discussion: Our study elucidates the critical role of ferroptosis in IDD and identifies specific genes, such as MT1G and CA9, as potential targets for diagnosis and therapy. These findings offer new insights into the molecular mechanisms underlying IDD and present promising avenues for future research and clinical applications.

Thrombospondin-1 promotes mechanical stress-mediated ligamentum flavum hypertrophy through the TGFß1/Smad3 signaling pathway.

Lumbar spinal canal stenosis is primarily caused by ligamentum flavum hypertrophy (LFH), which is a significant pathological factor. Nevertheless, the precise molecular basis for the development of LFH remains uncertain. The current investigation observed a notable increase in thrombospondin-1 (THBS1) expression in LFH through proteomics analysis and single-cell RNA-sequencing analysis of clinical ligamentum flavum specimens. In laboratory experiments, it was demonstrated that THBS1 triggered the activation of Smad3 signaling induced by transforming growth factor ß1 (TGFß1), leading to the subsequent enhancement of COL1A2 and α-SMA, which are fibrosis markers. Furthermore, experiments conducted on a bipedal standing mouse model revealed that THBS1 played a crucial role in the development of LFH. Sestrin2 (SESN2) acted as a stress-responsive protein that suppressed the expression of THBS1, thus averting the progression of fibrosis in ligamentum flavum (LF) cells. To summarize, these results indicate that mechanical overloading causes an increase in THBS1 production, which triggers the TGFß1/Smad3 signaling pathway and ultimately results in the development of LFH. Targeting the suppression of THBS1 expression may present a novel approach for the treatment of LFH.

Revealing the novel autophagy-related genes for ligamentum flavum hypertrophy in patients and mice model.

Background: Fibrosis is a core pathological factor of ligamentum flavum hypertrophy (LFH) resulting in degenerative lumbar spinal stenosis. Autophagy plays a vital role in multi-organ fibrosis. However, autophagy has not been reported to be involved in the pathogenesis of LFH. Methods: The LFH microarray data set GSE113212, derived from Gene Expression Omnibus, was analyzed to obtain differentially expressed genes (DEGs). Potential autophagy-related genes (ARGs) were obtained with the human autophagy regulator database. Functional analyses including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were conducted to elucidate the underlying biological pathways of autophagy regulating LFH. Protein-protein interaction (PPI) network analyses was used to obtain hub ARGs. Using transmission electron microscopy, quantitative RT-PCR, Western blotting, and immunohistochemistry, we identified six hub ARGs in clinical specimens and bipedal standing (BS) mouse model. Results: A total of 70 potential differentially expressed ARGs were screened, including 50 up-regulated and 20 down-regulated genes. According to GO enrichment and KEGG analyses, differentially expressed ARGs were mainly enriched in autophagy-related enrichment terms and signaling pathways related to autophagy. GSEA and GSVA results revealed the potential mechanisms by demonstrating the signaling pathways and biological processes closely related to LFH. Based on PPI network analysis, 14 hub ARGs were identified. Using transmission electron microscopy, we observed the autophagy process in LF tissues for the first time. Quantitative RT-PCR, Western blotting, and immunohistochemistry results indicated that the mRNA and protein expression levels of FN1, TGFß1, NGF, and HMOX1 significantly higher both in human and mouse with LFH, while the mRNA and protein expression levels of CAT and SIRT1 were significantly decreased. Conclusion: Based on bioinformatics analysis and further experimental validation in clinical specimens and the BS mouse model, six potential ARGs including FN1, TGFß1, NGF, HMOX1, CAT, and SIRT1 were found to participate in the fibrosis process of LFH through autophagy and play an essential role in its molecular mechanism. These potential genes may serve as specific therapeutic molecular targets in the treatment of LFH.

EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-ß1/Smad3 Signaling Pathway.

Background: The most common spinal disorder in elderly is lumbar spinal canal stenosis (LSCS). Previous studies showed that ligamentum flavum hypertrophy (LFH) with fibrosis as the main pathological change is one of the pathogenic factors leading to LSCS. Epidermal Growth Factor (EGF) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding EGF in LFH. The effect of EGF on the development of LFH is unknown, and the underlying pathomechanism remains unclear. In this study, we investigated the role of EGF in LFH and its potential molecular mechanism. Methods: First, the expression levels of EGF, phosphorylation of EGF receptor (pEGFR), Transforming growth factor-ß1 (TGF-ß1), Phosphorylated Smad3 (pSmad3), collagen I and collagen III were examined via immunohistochemistry and Western blot in LF tissues from patients with LSCS or Non-LSCS. Second, primary LF cells were isolated from adults with normal LF thickness and were cultured with different concentrations of exogenous EGF with or without erlotinib/TGF-ß1-neutralizing antibody. Results: The results showed that EGF, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression in the LSCS group was significantly higher than that in the Non-LSCS group. Meanwhile, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression was significantly enhanced in LF cells after exogenous EGF exposure, which can be notably blocked by erlotinib. In addition, pSmad3, collagen I and collagen III protein expression was blocked by TGF-ß1-neutralizing antibody. Conclusions: EGF promotes the synthesis of collagen I and collagen III via the TGF-ß1/Smad3 signaling pathway, which eventually contributes to LFH.

SMARCC2 combined with c­Myc inhibits the migration and invasion of glioma cells via modulation of the Wnt/ß­catenin signaling pathway.

Glioma is the most common type of central nervous system tumor. SWItch/sucrose non­fermentable (SWI/SNF) is a tumor suppressor that serves an important role in epithelial­mesenchymal transition (EMT). The present study aimed to identify key molecules involved in the EMT process. SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily c member 2 (SMARCC2) is mutated in and its expression is low in multiple types of cancer. SMARCC2 is the core subunit of the chromatin­remodeling complex, SWI/SNF. Relative mRNA SMARCC2 expression levels in human glioma tissue were analyzed via reverse transcription­quantitative PCR, whereas the protein expression levels were determined via immunohistochemistry staining. SMARCC2 expression was knocked down in glioma cells using small interfering RNA (si) and overexpressed by infection with adenovirus vectors carrying SMARCC2 cDNA. Wound healing and Transwell assays were performed to assess cell migration and invasion, respectively. Subsequently, immunofluorescence and western blotting were performed to analyze the expression levels of the oncogene c­Myc, which is associated with SMARCC2. SMARCC2 combines with C­MYC to downregulate its expression. Consistent with the results of the bioinformatics analysis, which revealed that the upregulated expression levels of SMARCC2 were associated with a more favorable prognosis in patients with glioma, the mRNA and protein expression levels of SMARCC2 were significantly upregulated in low­grade glioma tissues compared with high­grade glioma tissues. The results of the wound healing assay demonstrated that cell migration was significantly increased in the siSMARCC2­1/3 groups compared with the negative control (NC) group. By contrast, the migratory ability of cells was significantly reduced following transduction with adenovirus overexpressing SMARCC2, which upregulated the expression of SMARCC2, compared with the lentiviral vector­non­specific control (LVS­NC) group. The Transwell assay results further showed that SMARCC2 overexpression significantly inhibited the migratory and invasive abilities of U87MG and LN229 cells compared with the LVS­NC group. Co­immunoprecipitation assays were subsequently conducted to validate the binding of SMARCC2 and c­Myc; the results demonstrated that the expression of c­Myc was downregulated in adenovirus­transfected cells compared with LVS­NC­transfected cells. The results of the western blotting experiments demonstrated that the expression levels of N­cadherin, vimentin, snail family transcriptional repressor 1 and ß­catenin were notably downregulated, whereas the expression levels of T­cadherin were markedly upregulated in cell lines stably overexpressing SMARCC2 compared with the LVS­NC group. In conclusion, the results of the present study suggested that SMARCC2 may inhibit Wnt/ß­catenin signaling by regulating c­Myc expression in glioma. SMARCC2 regulates the EMT status of the glioblastoma cell line by mediating the expression of the oncogene C­MYC to inhibit its migration and invasion ability. Thus, SMARCC2 may function as a tumor suppressor or oncogene by regulating associated oncogenes or tumor suppressor genes.