Overexpression of RAD54L attenuates osteoarthritis by suppressing the HIF-1α/VEGF signaling pathway: Bioinformatics analysis and experimental validation

Abstract

Osteoarthritis (OA) is a widespread chronic, progressive, degenerative joint disease that causes pain and disability. Current treatments for OA have limited effectiveness and new biomarkers need to be identified. Bioinformatics analysis was conducted to explore differentially expressed genes and DNA repair/recombination protein 54 L (RAD54L) was selected. We firstly overexpressed RAD54L in interleukin-1β (IL-1β)-induced human articular chondrocytes or in OA rats to investigate its effect on OA. Chondrocyte viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Then we evaluated OA severity in vivo by Hematoxylin-eosin staining and Osteoarthritis Research Society International standards. The expression of inflammatory mediators was tested by enzyme-linked immunosorbent assay. Finally, western blot was performed to determine the relative expression level of hypoxia-inducible factors 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Overexpression of RAD54L promoted cell viability and attenuated apoptosis in IL-1β-induced human chondrocytes. A lower Osteoarthritis Research Society International score and a remarkable alleviation of chondrocyte disordering and infiltration of inflammatory cells were found in cartilage tissues of OA rats after overexpressing RAD54L. The inflammatory response induced by OA was decreased by RAD54L overexpression in vitro and in vivo. In addition, RAD54L overexpression decreased the relative expression level of HIF-1α and VEGF. Overexpression of RAD54L could attenuate OA by suppressing the HIF-1α/VEGF signaling pathway, indicating that RAD54L may be a potential treatment target for OA.

Fig 1. Identification of differentially expressed genes (DEGs).

(A) and (B) The volcano maps of DEGs in GSE98918 and GSE51588 respectively; red points presented up-regulated DEGs, and blue points presented down-regulated DEGs. (C) and (D) The box line plots showed the data correction results of the selected samples in GSE98918 and GSE51588 respectively. (E) and (F) The top 3 pathways of gene set enrichment analysis of DEGs in GSE98918 and GSE51588 respectively.

Fig 2. Heatmaps of common DEGs (co-DEGs).

(A) and (B) The heatmaps of co-DEGs in GSE98918 and GSE51588 respectively. Reprinted from [28] under a CC BY license, with permission from https://www.bioinformatics.com.cn, original copyright 2023.

Fig 3. Functional enrichment analysis of co-DEGs.

(A) A histogram and (B) A bubble diagram of Gene Ontology enrichment analysis of co-DEGs. (C) A histogram and (D) A bubble diagram of the Kyoto Encyclopedia of Genes and Genomes pathway analysis co-DEGs.

Fig 4. Hub gene analysis.

(A) A box line plot showed the expression difference between the Control group and the OA group in GSE98918. (B) A ridge diagram of hub genes. The horizontal axis stands for the gene expression, the shape of the peak stands for the dispersion among data, and the height stands for the number of samples corresponding to the gene expression. (C) A chordal graph with the expression changes of hub genes involved in the GO terms. Reprinted from [28] under a CC BY license, with permission from https://www.bioinformatics.com.cn, original copyright 2023. (D) Matrix correlation analysis chart. (E) Principal component analysis of hub genes.

Fig 5. The expression of hub genes in osteoarthritis (OA) model rats.

(A) The results of hematoxylin-eosin staining (200×, scale bars = 100 μm) and the inflammation score. (B) The Osteoarthritis Research Society International score. (C) The levels of proinflammatory cytokines (TNF-α, IL-6, and IL-18) in serum. (D) The relative mRNA expression of RAD54L, HJURP, UBE2C, CCNB2, SMC4, and CENPN. ** P < 0.01 vs. the Control group.

Fig 6. RAD54L overexpression promotes cell viability and inhibits the inflammatory response and apoptosis of IL-1β-induced human chondrocytes.

(A) RAD54L expression was detected by RT-qPCR. (B) Cell viability was detected by CCK-8 in chondrocytes. (C) The levels of proinflammatory cytokines (IL-6, IL-18, and TNF-α). (D) Flow cytometry was used to detect the apoptotic rates of chondrocytes. **P < 0.01 vs. the Control group. ^## P < 0.01 vs. the IL-1β group.

Fig 7. RAD54L overexpression attenuates osteoarthritis in rats.

(A) The protein expression level of RAD54L. (B) The relative mRNA expression level of RAD54L. (C) The results of hematoxylin-eosin staining (200×, scale bars = 100 μm) and the inflammation score. (D) The immunohistochemical staining of cleaved-caspase-3 (100× or 200×, scale bars = 200 or 100 μm). (E) The Osteoarthritis Research Society International score. (F) The levels of proinflammatory cytokines (TNF-α, IL-6, and IL-18) in serum. ** P < 0.01 vs. the Control group. ^## P < 0.01 vs. the IL-1β group.

Fig 8. The relative protein expression of HIF-1α and VEGF.

** P < 0.01 vs. the Control group. ^## P < 0.01 vs. the IL-1β group.