YTHDC1 Regulates the Migration, Invasion, Proliferation, and Apoptosis of Rheumatoid Fibroblast-Like Synoviocytes

Abstract

Background: Rheumatoid arthritis (RA), a chronic autoimmune condition, is characterized by persistent synovial inflammation, bone degradation, and progressive joint deterioration. Despite considerable research efforts, the precise molecular mechanism underlying RA remains elusive. This investigation aims to elucidate the potential role and molecular mechanism of N6-methyladenosine (m⁶A) methylation regulators in the pathogenesis of RA.

Methods: In this study, we employed bioinformatics tools to elucidate the association between RA and m⁶A modifications, aiming to identify potential biological markers. We extracted datasets GSE12021, GSE55235, and GSE55457 from the Gene Expression Omnibus (GEO) database for comprehensive analysis. Utilizing differential expression analysis, protein-protein interaction (PPI) analysis, and single-cell sequencing techniques, we identified pivotal hub genes implicated in the pathogenesis of RA. Subsequently, we assessed the correlation between these hub genes and the pathogenesis of RA using Gene Set Enrichment Analysis (GSEA). Both in vivo and in vitro experiments were performed to confirm the expression and functional roles of the identified key hub gene in RA.

Results: Differential expression analysis, PPI analysis, and single-cell analysis identified three key hub genes (YTHDC1, YTHDC2, and YTHDF2) associated with RA. GSEA results further revealed that these genes are enriched in pathways associated with inflammatory responses. Subsequent correlation analysis demonstrated a significant negative correlation between YTHDC1 expression and CD8+ T cell levels. Notably, the gene and protein expression levels of YTHDC1 and YTHDF2 were significantly reduced in the synovial tissue of RA patients. Furthermore, silencing YTHDC1 in fibroblast-like synoviocytes (FLSs) significantly inhibited their migration, invasion, proliferation, and induced apoptosis.

Conclusion: YTHDC1 may potentially be involved in the pathogenesis of RA through its regulation of migration, invasion, proliferation, and apoptosis in FLSs.

Keywords: M6A modification; YTHDC1; apoptosis; proliferation; rheumatoid arthritis.

Figure 1

Identification of DEGs in synovial tissue from patients with RA included: (A) Results of principal component analysis (PCA) before batch effect removal. (B) PCA results following batch effect removal. (C) Volcano plot representing all normalized mRNA expression data. (D) Heatmap illustrating DEGs in synovial tissue from RA patients, with criteria of |log FC| > 1 and P-value < 0.05.

Figure 2

Enrichment analysis and immune infiltration analysis included: (A) KEGG enrichment analysis of DEGs with a q-value < 0.05. (B) GO enrichment analysis of DEGs with a P-value < 0.05. (C) Analysis of differences in immune cell infiltration, with significance defined as P-value < 0.05. statistical significance denoted as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Screening of hub genes involved the following steps: (A) Quality control chart showing single-cell data before filtering, including all cells to provide a full overview of the dataset. (B) Results from cluster analysis of single-cell data. (C) Hub genes were identified from the PPI network using the MCODE algorithm. (D) Hub genes were extracted from the PPI network via the Degree algorithm. (E) Venn diagram illustrating the overlap of key genes.

Figure 4

Correlation analysis between hub genes and RA. Gene Set Enrichment Analysis (GSEA) included: (A) Pathway enrichment analysis for YTHDC1. (B) Pathway enrichment analysis for YTHDC2. (C) Pathway enrichment analysis for YTHDF2. (D) Analysis of the correlation between key genes and immune cell infiltration. (E) Hub genes in the merged dataset were analyzed using ROC curves.

Figure 5

Expression of hub genes in synovial tissue from patients with RA. (A) Representative MRI scans. (B) Histological evaluations of H&E-stained synovial tissue sections (n = 3). (C) m⁶A modification levels quantified via qRT-PCR, normalized against GAPDH (n = 10). (D) mRNA expression levels of YTHDC1, YTHDC2, and YTHDF2 in TC and RA samples (n = 9). (E) Protein concentrations of YTHDC1, YTHDC2, and YTHDF2 in TC and RA samples (n = 3). (F) IHC images showing the expression of YTHDC1 and YTHDF2 in TC and RA samples (n = 3). (G) IF staining displaying YTHDC1 and YTHDF2 in TC and RA samples, with DAPI (blue), VIMENTIN (red), YTHDC1/YTHDF2 (green), and merged images (n = 3). Data are presented as means ± SD; significance levels are indicated as ns (not significant), ***P < 0.001.

Figure 6

YTHDC1 modulates apoptosis in RA-FLSs. (A, B) Overexpression of YTHDC1 in RA-FLSs was performed, with efficacy confirmed via (A) quantitative RT-PCR and (B) Western blot analysis. (C) Western blotting assessed the protein levels of Bcl-2, Bax, Caspase-3, and Cleaved-caspase-3 subsequent to YTHDC1 overexpression. (D) Apoptosis in RA-FLSs following YTHDC1 overexpression was quantified using flow cytometry with 7-AAD and Annexin V staining (n = 3). (E) Apoptotic cells were further evaluated by TUNEL staining, and the number of TUNEL-positive cells was quantified (n = 3). Data are presented as means ± SD; significance levels are indicated as ns (not significant), **P < 0.01, ***P < 0.001.

Figure 7

YTHDC1 influences the migration, invasion, and proliferation of RA-FLSs. (A) A representative photomicrograph and a column chart displaying EdU assay results (n = 3) are shown. (B) Western blot analysis was utilized to assess the protein levels of PCNA, CDK4, and Cyclin D1 following YTHDC1 overexpression. (C, D) The impact of YTHDC1 on the migratory capacity of RA-FLSs was evaluated using a (C) wound healing assay (n = 3) and (D) a Transwell migration assay (n = 3). (E) The invasive potential of RA-FLSs was quantified using a Matrigel-coated Transwell assay (n = 3). (F, G) YTHDC1 knockdown was performed in FLSs, with the efficacy confirmed via quantitative RT-PCR and Western blot analysis. (H) Following overexpression of YTHDC1, protein expression levels of PCNA, Bcl-2, and Bax were determined by Western blot. Data are presented as means ± SD; statistical significance denoted as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

The diagram depicts the regulatory effect of YTHDC1 on the activities of Fibroblast-Like Synoviocytes (FLSs) in the context of rheumatoid arthritis (RA), encompassing their migration, invasion, proliferation, and apoptosis processes. As RA advances, hyperplasia and thickening of the synovial tissue occur. In parallel, enhanced expression of YTHDC1 mitigates FLS activity by influencing the expression of critical proteins including PCNA, CDK4, Cyclin D1, Bcl2, Bax, Caspase3, and Cleaved-caspase3.