Identification of circ-FAM169A sponges miR-583 involved in the regulation of intervertebral disc degeneration

Abstract

Objective: Low back pain (LBP) is the predominant cause of disc degeneration in patients, which brings serious social problems and economic burdens. Increasing evidence has indicated that intervertebral disc degeneration (IDD) is one of the most common causes triggering LBP. Accumulating evidence has shown that circRNAs are involved in the pathological process of IDD. Nevertheless, the circRNA-mediated IDD pathogenesis still remains unknown. This study explored the potential mechanism and functions of circ-FAM169A in NPCs.

Methods: Bioinformatics analysis was conducted to identify key circRNA, miRNA and mRNA and predict their potential role in IDD. Dual-luciferase reporter assay, western blot, qRT-PCR, and fluorescence in situ hybridisation (FISH) were used to demonstrate the interaction among circ-FAM169A, miR-583 and Sox9 in NPCs.

Results: Herein, we identified circ-FAM169A, which was dramatically up-regulated in degenerative nucleus pulposus (NP) tissues and negatively correlated with expression levels of miR-583. We constructed a circ-FAM169A-miR-583-mRNAs co-expression network and predicted circ-FAM169A-miR-583 pathway predominantly involved in extracellular matrix metabolism and cell apoptosis etc. FISH experiments confirmed circ-FAM169A and miR-583 co-existence in the cytoplasm of NPCs. Luciferase reporter assay illustrated that circ-FAM169A was directly bound to miR-583 and Sox9 was the directly target gene of miR-583. Additionally, miR-583 negatively regulated Sox9 mRNA and protein levels in NPCs.

Conclusion: Findings of this study indicated that circ-FAM169A-miR-583 pathway may play a significant role in the regulation of IDD, which will provide novel diagnostic biomarkers and develop effective treatment strategy of IDD diseases.

The translational potential of this article: This study suggested that circ-FAM169A-miR-583 pathway may regulate NPCs apoptosis and extracellular matrix synthesis and catabolism by targeting Sox9. It provides a novel therapeutic target and strategy for IVDD diseases.

Keywords: Competitive endogenous RNA; Differential expressed circRNAs; GEO; Intervertebral disc degeneration; circ-FAM169A; miR-583.

Figure 1

Identification of GSE67566 dataset from GEO. (A) The corplot_heatmap of 5 normal NP tissues and 5 degenerative NP tissues. (B) The box plot was used to assess the distributions of DECs intensities in all the degenerative and normal tissues. (C) The length distribution of DECs. (D) The distribution of DECs in all human chromosomals. (E) Hierarchical cluster analysis of the 49 DECs in IDD on the basis of GSE67566, revealing that 19 circRNAs were up-regulated whereas 30 circRNAs were down-regulated, with rows representing circRNAs and columns representing tissues. The color scale varies from red to blue; red points illustrate up-regulated circRNAs and blue points illustrate down-regulated circRNAs. (F) The scatter plot for DECs in IDD on the basis of GSE67566, with red points representing up-regulation and green points representing down-regulation. GEO, Gene Expression Omnibus; DECs, differential expressed circRNAs; circRNA, circular RNA; IDD, intervertebral disc degeneration.

Figure 2

Prediction of circ-FAM169A target miRNAs and validation of their expression in IDD. (A) qRT-PCR assay detected the expression of circ-FAM169A in 15 human normal NP tissues and 15 human degenerative NP tissues (∗∗P ​< ​0.01 by unpaired Student’s t-test). (B) Circ-FAM169A target miRNAs were predicted using Venn diagram. (C) The expression of miR-583 was remarkably down-regulated in degenerative NP tissues (∗∗P ​< ​0.01 by unpaired Student’s t-test). (D) qRT-PCR assay determined the expression of miR-583 in response to IL-1β and TNF-α stimulation in NPCs (∗P ​< ​0.05 by unpaired Student’s t-test). Data were shown as the mean ​± ​SEM of three independent experiments. miRNAs, microRNAs; qRT-PCR, quantitative real-time polymerase chain reaction; NP, nucleus pulposus; IL-1β, interleukin -1β; TNF-α, tumour necrosis factor-α.

Figure 3

Circ-FAM169A interaction with miR-583. (A) circ-FAM169A and miR-583 binding sites. (B) The wild and mutant sequences of circ-FAM169A. (C) Dual-luciferase reporter assay was performed to detect the activity of LUC-circ-FAM169A or LUC-circ-FAM169A-mut1/2 in NPCs co-transfected with miR-583 mimic or inhibitor. (∗∗P ​< ​0.01 by one-way ANOVA followed by Turkey’s multiple comparisons test). (D) FISH showed that circ-FAM169A colocalized with miR-583 in the cytoplasm. Nuclei were stained with DAPI. Circ-FAM169A, miR-583 and nuclei were labeled with red, green and blue, respectively. Scale bar ​= ​20 ​μm. Data were shown as the mean ​± ​SEM of three independent experiments. NPCs, nucleus pulposus cells; FISH, Fluorescence in situ hybridisation; DAPI, 4,6-diamidino-2-phenylindole.

Figure 4

Circ-FAM169A repressed the expression of miR-583 in NPCs. (A) Construction of the over-expression vector of circ-FAM169A. (B) qRT-PCR assay corroborated that the expression of circ-FAM169A was significantly up-regulated by circ-FAM169A OE in NPCs (∗∗∗P ​< ​0.001 by unpaired Student’s t-test). (C) The expression of miR-583 was repressed by circ-FAM169A OE in NPCs using qRT-PCR (∗∗P ​< ​0.01 by unpaired Student’s t-test). Data were shown as the mean ​± ​SEM of three independent experiments. NPCs, nucleus pulposus cells; qRT-PCR, quantitative real-time polymerase chain reaction; OE, over-expression.

Figure 5

Functional enrichment analysis of the parental genes of DECs. (A) R software clusterProfiler package was employed to analyze KEGG pathway. Statistical significance was set up at p values of <0.05. (B) The Reactome pathway analysis of the parental genes of DECs. DECs, differential expressed circRNAs; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

The pathway analysis of miR-583 target genes. (A) Venn diagram was used to select the overlapping target genes of miR-583 in TargetScan, miRDB, mirDIP and miRWalk databases. (B) The circ-FAM169A-miR-583-mRNAs network was constructed based on the overlapping mRNAs. Circ-FAM169A, miR-583, miR-583 target mRNAs and IDD-related mRNAs were indicated by green diamonds, purple triangles, blue squares and red ellipse, respectively. (C) KEGG pathway analysis of the predicted target genes of miR-583. (D) Reactome pathway analysis was conducted to detect the potential biological functions of target mRNAs, and the results showed that at least 5 pathways have correlation with PI3K-AKT signaling pathway. (E) Top 5 enriched GO terms of miR-583 target genes. The X-axis represents the GO term, and the Y-axis represents the gene ratio. The red, green and blue columns indicate the biological process, cellular component, and molecular function, respectively. circRNA, circular RNA; miRNA, microRNA; mRNA, messenger RNA; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.

Figure 7

miR-583 was directly bound to the 3′-UTR of Sox9. (A) The binding site of miR-583 and Sox9 as well as the WT and mut sequences of Sox9. (B) Dual-luciferase reporter assay demonstrated that miR-583 mimic inhibited the luciferase activity of Sox9-WT vector and miR-583 inhibitor elevated the activity in NPCs, whereas such over-expression and inhibition could not alter the activity of Sox9-mut vector after co-transfected with miR-583 mimic or inhibitor. (∗∗P ​< ​0.01 by one-way ANOVA followed by Turkey’s multiple comparisons test). (C) qRT-PCR assay demonstrated that miR-583 can negatively regulate Sox9 mRNA level in NPCs. (∗P ​< ​0.05 and ∗∗∗P ​< ​0.001 by one-way ANOVA followed by Turkey’s multiple comparisons test). (D) The protein expression level of Sox9 in NPCs was detected using WB assay. Data were shown as the mean ​± ​SEM of three independent experiments. Sox9, SRY-related high mobility group box 9; NPCs, nucleus pulposus cells.