Long noncoding and micro-RNA expression in a model of articular chondrocyte degeneration induced by stromal cell-derived factor-1

Abstract

Background: Gene regulatory network analysis has found that long noncoding ribonucleic acids (lncRNAs) are strongly associated with the pathogenesis of osteoarthritis.

Objectives: To determine the differential expression of lncRNAs and microRNAs (miRNAs) in normal chondrocytes and those from a model of articular chondrocyte degeneration.

Methods: Chondrocytes were cultured from cartilage obtained from patients diagnosed with osteoarthritis of the knee. Stromal cell-derived factor-1 (SDF-1) was used to induce their degeneration. Total RNA was extracted, analyzed, amplified, labeled, and hybridized on a chip to determine expression. The set of enriched differentially expressed miRNAs was analyzed by gene ontology and the Kyoto Encyclopedia of Genes and Genomes to describe the functional properties of the key biological processes and pathways. We conducted a bioinformatics analysis using Cytoscape to elucidate the interactions between miRNAs and proteins.

Results: We found that the expression of 186 lncRNAs was significantly different in the model of chondrocyte degeneration, in which 88 lncRNAs were upregulated, and 98 were downregulated. Expression of 684 miRNAs was significantly different. Analysis of the protein-protein interaction (PPI) network indicated that the genes for CXCL10, ISG15, MYC, MX1, OASL, IFIT1, RSAD2, MX2, IFI44L, and BST2 are the top 10 core genes, identifying the most important functional modules to elucidate the differential expression of miRNAs.

Conclusions: These data may provide new insights into the molecular mechanisms of chondrocyte degeneration in osteoarthritis, and the identification of lncRNAs and miRNAs may provide potential targets for the differential diagnosis and therapy of osteoarthritis.

Keywords: RNA; chondrocytes; high-throughput nucleotide sequencing; long noncoding; microRNAs; osteoarthritis.

Figure 1

Morphology of chondrocytes in the 2 groups (×100). A. Chondrocytes in the experimental group were cultured with SDF-1 for 48 h. B. Morphology of chondrocytes in the control group. SDF-1, stromal cell-derived factor-1.

Figure 2

Analysis of miRNAs and lncRNAs. A. Heatmap of the differentially expressed (DE) miRNAs. B. Scatter plot of the DE miRNAs. C. Volcano plot of the DE miRNAs. D. Heatmap of the DE lncRNAs. E. Scatter plot of the DE lncRNAs. F. Volcano plot of the DE lncRNAs. In the heatmap, red represents upregulated miRNAs or lncRNAs, and green represents downregulated miRNAs or lncRNAs. In the scatter plot, the X and Y values are the average normalized signal values, shown on a log₂ scale. The red and green lines were set as FC lines with a default change of 2.0. Red points (FC >2) indicate upregulated miRNAs or lncRNAs, and blue points (FC ≤2) indicate downregulated miRNAs or lncRNAs. In the volcano plot, the X-axis is the FC (log₂), and the Y-axis is P (−log₁₀). Red points (FC >2) indicate upregulated miRNAs or lncRNAs, and blue points (FC ≤2) indicate downregulated miRNAs or lncRNAs. DE, differentially expressed; FC, fold change; FPKM, fragments per kilobase of transcript per million mapped fragments; lncRNAs, long noncoding ribonucleic acids; miRNAs, microRNAs; SDF1, stromal cell-derived factor-1 treated articular chondrocytes. Normal indicates articular chondrocytes untreated with SDF-1.

Figure 3

Expression signatures of dysregulated lncRNAs in SDF-1-induced articular chondrocyte degeneration. A. Length distribution showed that dysregulated lncRNAs were mainly concentrated between 700 bp and 3000 bp. B. Differential lncRNAs were classified according to their genomic architecture.

Figure 4

GO analysis of differentially expressed genes. GO annotations of mRNAs with top 30 enrichment scores. The circles represent biological processes; the triangles represent cell components; and the squares represent MF. GO, gene ontogeny; MF, molecular functions.

Figure 5

KEGG signaling pathway analysis of differentially expressed genes. Top 30 for KEGG enrichment. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

PPI network analysis of the top 10 differentially expressed genes. Nodes represent genes for the proteins indicated. Lines indicate interactions between genes. Red indicates upregulated genes, and green indicates downregulated genes. PPI, protein–protein interaction.