Identification of SLC3A2 as a Potential Therapeutic Target of Osteoarthritis Involved in Ferroptosis by Integrating Bioinformatics, Clinical Factors and Experiments

Abstract

Osteoarthritis (OA) is a type of arthritis that causes joint pain and limited mobility. In recent years, some studies have shown that the pathological process of OA chondrocytes is related to ferroptosis. Our study aims to identify and validate differentially expressed ferroptosis-related genes (DEFRGs) in OA chondrocytes and to investigate the potential molecular mechanisms. RNA-sequencing and microarray datasets were downloaded from Gene Expression Omnibus (GEO) data repository. Differentially expressed genes (DEGs) were screened by four methods: limma-voom, edgeR, DESeq2, and Wilcoxon rank-sum test. Weighted correlation network analysis (WGCNA), protein-protein interactions (PPI), and cytoHubba of Cytoscape were applied to identify hub genes. Clinical OA cartilage specimens were collected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, western blotting (WB), histological staining, transmission electron microscopy (TEM), and transfection. Sankey diagram was used to visualize the relationships between the expression level of SLC3A2 in the damaged area and clinical factors. Based on bioinformatics analysis, clinical factors, and experiment validation, SLC3A2 was identified as a hub gene. It was down-regulated in OA cartilage compared to normal cartilage (p < 0.05). Functional enrichment analysis revealed that SLC3A2 was associated with ferroptosis-related functions. Spearman correlation analysis showed that the expression level of SLC3A2 in the OA cartilage-damaged area was closely related to BMI, obesity grade, and Kellgren-Lawrence grade. Furthermore, in vitro experiments validated that SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. In summary, we demonstrated that SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. These findings provide a new idea for the study of the pathogenesis of OA, thus providing new means for the clinical diagnosis and targeted therapy of OA.

Keywords: GEO; cartilage; ferroptosis; osteoarthritis.

Figure 1

The flowchart of this research. All data were presented as the mean ± SEM: **** p < 0.0001.

Figure 2

Data preprocessing and identification of DEGs. PCA analyses were performed to visualize the effect before (a) and after (b) removing the batch effect. Boxplots were used to show data before (c) and after (d) TMM normalization. (e) Differential genes were obtained by four algorithms. (f) A Venn diagram was used to obtain the intersection genes of the four methods.

Figure 3

Functional enrichment and PPI of DEFRGs in the normal and OA samples. (a,b) The results of the GO analysis were displayed in lollipop and circle charts. (c,d) The results of the KEGG analysis were displayed in tree maps and circle charts.

Figure 4

Weighted correlation network analysis. (a) Dendrogram of expressed genes in the top 10,000 of MAD clustered based on a dissimilarity measure (1  −  TOM). (b) Correlation between OA trait and modules. (c,d) A scatterplot of Gene Significance (GS) vs. Module Membership (MM) in the module. There is a highly significant correlation between GS and MM in blue and cyan modules.

Figure 5

Identification and validation of SLC3A2. (a) Validation of differential expression of SLC3A2 in OA and normal samples by qRT-PCR (n = 6). (c,d) Western blotting and quantification analysis in OA and normal samples (n = 6). (e) Alcian blue, safranin O, and toluidine blue staining. (b,f,g) qRT-PCR and WB analysis in undamaged(U) and damaged (D) areas of OA cartilage(n = 6). All data were presented as the mean ± SEM: * p < 0.05, and **** p < 0.0001.

Figure 6

Relationship between SLC3A2 expression and clinical factors. (a) Heatmaps of relative RNA expression of SLC3A2 in undamaged (U) and damaged (D) areas (n = 30). (b,c) Sankey diagram presents the relationship between age, gender, obesity grade, Kellgren-Lawrence, and expression of SLC3A2 in the damaged area. All data were expressed as the mean ± SEM: **** p < 0.0001.

Figure 7

Functional experiments of SLC3A2. (a) The efficacy of SLC3A2-specific shRNA was measured by qRT-PCR in chondrocytes. (b,c) Protein expression of COL2A1, MMP13 in chondrocytes transfected with sh-NC and sh-SLC3A2 analyzed by WB. (d) Mitochondrial morphology of chondrocytes transfected with sh-NC and sh-SLC3A2 was observed using transmission electron microscopy (n = 3). (e,f) Fer-1 decreased the MMP13 expression and increased the COL2A1 expression in the transfected chondrocytes. All data were expressed as the mean ± SEM: * p < 0.05, *** p < 0.001, **** p < 0.0001, and ^## p > 0.05.