Dataset Information

Construction of circ_0071922-miR-15a-5p-mRNA network in intervertebral disc degeneration by RNA-sequencing.

ABSTRACT:

Background

Low back pain (LBP) is the main factor of global disease burden. Intervertebral disc degeneration (IVDD) has long been known as the leading reason of LBP. Increasing studies have verified that circular RNAs (circRNAs)-microRNAs (miRNAs)-mRNAs network is widely involved in the pathological processes of IVDD. However, no study was made to demonstrate the circRNAs-mediated ferroptosis, oxidative stress, extracellular matrix metabolism, and immune response in IVDD.

Methods

We collected 3 normal and 3 degenerative nucleus pulposus tissues to conduct RNA-sequencing to identify the key circRNAs and miRNAs in IVDD. Bioinformatics analysis was then conducted to construct circRNAs-miRNAs-mRNAs interaction network associated with ferroptosis, oxidative stress, extracellular matrix metabolism, and immune response. We also performed animal experiments to validate the therapeutic effects of key circRNAs in IVDD.

Results

We found that circ_0015435 was most obviously upregulated and circ_0071922 was most obviously downregulated in IVDD using RNA-sequencing. Then we observed that hsa-miR-15a-5p was the key downstream of circ_0071922, and hsa-miR-15a-5p was the top upregulated miRNA in IVDD. Bioinformatics analysis was conducted to predict that 56 immunity-related genes, 29 ferroptosis-related genes, 23 oxidative stress-related genes and 8 ECM-related genes are the targets mRNAs of hsa-miR-15a-5p. Then we constructed a ceRNA network encompassing 24 circRNAs, 6 miRNAs, and 101 mRNAs. Additionally, we demonstrated that overexpression of circ_0071922 can alleviate IVDD progression in a rat model.

Conclusions

The findings of this study suggested that circ_0071922-miR-15a-5p-mRNA signaling network might affect IVDD by modulating the nucleus pulposus cells ferroptosis, oxidative stress, ECM metabolism, and immune response, which is an effective therapeutic targets of IVDD.

SUBMITTER: Li Y

PROVIDER: S-EPMC10782064 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Publications

Construction of circ_0071922-miR-15a-5p-mRNA network in intervertebral disc degeneration by RNA-sequencing.

Li Yongjin Y Wang Baobao B Sun Wenzhi W Kong Chao C Ding Junzhe J Hu Feng F Li Jianhua J Chen Xiaolong X Lu Shibao S

JOR spine 20230824 1

<h4>Background</h4>Low back pain (LBP) is the main factor of global disease burden. Intervertebral disc degeneration (IVDD) has long been known as the leading reason of LBP. Increasing studies have verified that circular RNAs (circRNAs)-microRNAs (miRNAs)-mRNAs network is widely involved in the pathological processes of IVDD. However, no study was made to demonstrate the circRNAs-mediated ferroptosis, oxidative stress, extracellular matrix metabolism, and immune response in IVDD.<h4>Methods</h4> ...[more]

PMID: 38222808

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Project description:Accumulating evidence has indicated that noncoding RNAs are involved in intervertebral disc degeneration (IDD); however, the competing endogenous RNA (ceRNA)‑mediated regulatory mechanisms in IDD remain rarely reported. The present study aimed to comprehensively investigate the alterations in expression levels of circular RNA (circRNA), long noncoding RNA (lncRNA), microRNA (miRNA/miR) and mRNA in the nucleus pulposus (NP) of patients with IDD. In addition, crucial lncRNA/circRNA‑miRNA‑mRNA ceRNA interaction axes were screened using the GSE67567 microarray dataset obtained from the Gene Expression Omnibus database. After data preprocessing, differentially expressed circRNAs (DECs), lncRNAs (DELs), miRNAs (DEMs) or genes (DEGs) between IDD and normal controls were identified using the Linear Models for Microarray data method. A protein‑protein interaction (PPI) network was constructed for DEGs based on protein databases, followed by module analysis. The ceRNA network was constructed based on the interaction between miRNAs and mRNAs, and lncRNAs/circRNAs and miRNAs. The underlying functions of mRNAs were predicted using the Database for Annotation, Visualization and Integrated Discovery database. The present study identified 636 DECs, 115 DELs, 84 DEMs and 1,040 DEGs between patients with IDD and control individuals. PPI network analysis demonstrated that Fos proto‑oncogene, AP‑1 transcription factor subunit (FOS), mitogen‑activated protein kinase 1 (MAPK1), hypoxia inducible factor 1 subunit α (HIF1A) and transforming growth factor β1 (TGFB1) were hub genes and enriched in modules. Metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1)/hsa_circRNA_102348‑hsa‑miR‑185‑5p‑TGFB1/FOS, MALAT1‑hsa‑miR‑155‑5p‑HIF1A, hsa_circRNA_102399‑hsa‑miR‑302a‑3p‑HIF1A, MALAT1‑hsa‑miR‑519d‑3p‑MAPK1 and hsa_circRNA_100086‑hsa‑miR‑509‑3p‑MAPK1 ceRNA axes were obtained by constructing the ceRNA networks. In conclusion, these identified ceRNA interaction axes may be crucial targets for the treatment of IDD.

Project description:BackgroundIntervertebral disc (IVD) degeneration is a multifactorial pathological process resulting in the dysregulation of IVD cell activity. The catabolic shift observed in IVD cells during degeneration leads to increased inflammation, extracellular matrix (ECM) degradation, aberrant intracellular signaling and cell loss. Importantly, these pathological processes are known to be interconnected and to collectively contribute to the progression of the disease. MicroRNAs (miRNAs) are known as strong post-transcriptional regulators, targeting multiple genes simultaneously and regulating numerous intracellular pathways. Specifically, miR-155-5p has been of particular interest since it is known as a pro-inflammatory mediator and contributing factor to diseases like cancer and osteoarthritis. This study investigated the role of miR-155-5p in IVD degeneration with a specific focus on inflammation and mechanosensing.MethodsGain- and loss-of-function studies were performed through transfection of human Nucleus pulposus (NP) and Annulus fibrosus (AF) cells isolated from degenerated IVDs with miR-155-5p mimics, inhibitors or their corresponding non-targeting control. Transfected cells were then subjected to an inflammatory environment or mechanical loading. Conditioned media and cell lysates were collected for phosphorylation and cytokine secretion arrays as well as gene expression analysis.ResultsIncreased expression of miR-155-5p in AF cells resulted in significant upregulation of interleukin (IL)-8 cytokine secretion during cyclic stretching and a similar trend in IL-6 secretion during inflammation. Furthermore, miR-155-5p mimics increased the expression of the brain-derived neurotrophic factor (BDNF) in AF cells undergoing cyclic stretching. In NP cells, miR-155-5p gain-of-function resulted in the activation of the mitogen-activated protein kinase (MAPK) signaling pathway through increased phosphorylation of p38 and p53. Lastly, miR-155-5p inhibition caused a significant increase in the anti-inflammatory cytokine IL-10 in AF cells and the tissue inhibitor of metalloproteinases (TIMP)-4 in NP cells respectively.ConclusionOverall, these results show that miR-155-5p contributes to IVD degeneration by enhancing inflammation through pro-inflammatory cytokines and MAPK signaling, as well as by promoting the catabolic shift of AF cells during mechanical loading. The inhibition of miR-155-5p may constitute a potential therapeutic approach for IVD degeneration and low back pain.

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.

Dataset Information

Construction of circ_0071922-miR-15a-5p-mRNA network in intervertebral disc degeneration by RNA-sequencing.

Background

Methods

Results

Conclusions

Publications

Construction of circ_0071922-miR-15a-5p-mRNA network in intervertebral disc degeneration by RNA-sequencing.

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