Dataset Information

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

ABSTRACT: Summary

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.

SUBMITTER: Li Y

PROVIDER: S-EPMC7773979 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Project description:ObjectiveLow back pain is a leading cause of disabilities worldwide, and intervertebral disc degeneration (IVDD)-related disorders have been recognised as one of the main contributors. Nevertheless, the underlying mechanism has not yet been fully understood. The aim of this study was to investigate the role of the miR-133a-5p/FBXO6 axis in the regulation of IVDD.MethodsRT-qPCR, WB and IHC were performed to assess the expression of FBXO6 in human IVD tissues. Nucleus pulposus (NP) cells were treated with IL-1β to induce IVDD cellular model. Silence of FBXO6 was achieved using specific siRNAs. CCK-8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to evaluate the role and mechanism of FBXO6 in the process of IVDD. Online tools, GSE datasets and RT-qPCR were used to search the candidate miRNAs targeting FBXO6. The direct binding sites between FBXO6 and miR-133a-5p were further verified by a dual luciferase assay. RT-qPCR, WB and rescue experiments were conducted to identify the regulatory function of miR-133a-5p on the expression of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2. In addition, the role of the NF-κB pathway in regulating miR-133a-5p was studied using lentiviral shRNA, WB and RT-qPCR.ResultsResults showed that FBXO6 mainly expressed in the NP tissue of IVD and the expression of FBXO6 decreased with the process of IVDD as well as under IL-1β stimulation. The silence of FBXO6 led to the decreased expression of aggrecan and collagen Ⅱ and the increased expression of MMP3, ADAMTS5, IL-6 and COX2, which further induced the degeneration of NP cells. The bioinformatic analysis showed that miR-133a-5p was the candidate miRNA targeting FBXO6. miR-133a-5p was upregulated in IVDD tissues and significantly inhibited the expression of FBXO6. The inhibition of miR-133a-5p ameliorated the acceleration of IVDD induced by the silence of FBXO6 in vitro. Moreover, it was demonstrated that IL-1β regulated the expression of the miR-133a-5p/FBXO6 axis via the NF-κB pathway in NP cells.ConclusionmiR-133a-5p was upregulated by IL-1β to aggravate intervertebral disc degeneration via sponging FBXO6. Inhibiting miR-133a-5p expression or rescuing FBXO6 expression may be promising strategies for the treatment of IVDD.The translational potential of this articleThis study suggests that the miR-133a-5p/FBXO6 axis could regulate NP cells proliferation, apoptosis, synthesis and degradation of extracellular matrix, which provides a promising therapeutic target and strategy for the treatment of IVDD.