Identification of transcription factors responsible for dysregulated networks in human osteoarthritis cartilage by global gene expression analysis

Abstract

Objective: Osteoarthritis (OA) is the most prevalent joint disease. As disease-modifying therapies are not available, novel therapeutic targets need to be discovered and prioritized for their importance in mediating the abnormal phenotype of cells in OA-affected joints. Here, we generated a genome-wide molecular profile of OA to elucidate regulatory mechanisms of OA pathogenesis and to identify possible therapeutic targets using integrative analysis of mRNA-sequencing data obtained from human knee cartilage.

Design: RNA-sequencing (RNA-seq) was performed on 18 normal and 20 OA human knee cartilage tissues. RNA-seq datasets were analysed to identify genes, pathways and regulatory networks that were dysregulated in OA.

Results: RNA-seq data analysis revealed 1332 differentially expressed (DE) genes between OA and non-OA samples, including known and novel transcription factors (TFs). Pathway analysis identified 15 significantly perturbed pathways in OA with ECM-related, PI3K-Akt, HIF-1, FoxO and circadian rhythm pathways being the most significantly dysregulated. We selected DE TFs that are enriched for regulating DE genes in OA and prioritized these TFs by creating a cartilage-specific interaction subnetwork. This analysis revealed eight TFs, including JUN, Early growth response (EGR)1, JUND, FOSL2, MYC, KLF4, RELA, and FOS that both target large numbers of dysregulated genes in OA and are themselves suppressed in OA.

Conclusions: We identified a novel subnetwork of dysregulated TFs that represent new mediators of abnormal gene expression and promising therapeutic targets in OA.

Keywords: Cartilage; Gene expression; Transcription factors.

Figure 1-

Overview of the data analysis.

Figure 2 -. Transcriptomic landscape of normal and OA knee articular cartilage.

A) Multidimensional scaling (MDS) plot of gene expression (lcpm) in normal (blue) and OA articular cartilage samples (red) reveals strong clustering of samples by phenotype. B) Unsupervised hierarchical clustering of normal and OA articular cartilage samples based on the expression levels of the top 1000 differentially expressed genes ranked by adjusted p-value. C) Volcano plot representation of gene expression analysis in normal and OA articular cartilage samples highlighting the most differentially expressed genes. D) Bar plot representing top results of gene ontology (GO) enrichment analysis of differentially expressed genes between normal and OA articular cartilage samples. E) Bar plot representing all 15 significantly enriched KEGG pathways in differentially expressed genes between normal and OA articular cartilage samples.

Figure 3 -. Transcription factor analysis.

A) Overlap between differentially expressed transcription factors and transcription factors enriched for binding sites in the promoter region of differentially expressed genes in normal and OA articular cartilage results in 14 DE enriched TFs. B) Heatmap of normal and OA articular cartilage samples based on the expression of 14 DE enriched transcription factors. C) Top 5 differentially expressed TFs with highest percentage of target genes that are differentially expressed in normal and OA articular cartilage.

Figure 4-. Network-based prioritization of differentially expressed genes in OA.

A) Network diagram of the 64 gene subnetwork from the HumanBase cartilage-specific network seeded with the 14 differentially expressed (DE) enriched TFs. Node shape represented by diamonds represent the 14 DE enriched TFs, node color indicates significantly up- (red) or down- (blue) regulated in OA vs Normal articular cartilage and node size corresponds to the degree (larger nodes have more connections to other nodes). Edges not drawn between nodes to aid in visualization of the membership of each gene within significantly enriched pathways, labeled with text and highlighted by background color. B) Unsupervised hierarchical clustering of normal and OA articular cartilage samples based on the expression levels of DE genes that belong to the HIF-1 signaling pathway, pathways cancer, and FoxO signaling pathway. C) Ranking of genes in the network based on their degree. DE genes are marked with an asterisk. TFs that are DE and enriched for binding to DE genes are highlighted in red.