Dataset Information

Mechanism of protective role of miR-874-3p in intervertebral disc degeneration.

ABSTRACT:

Background

The intervertebral disc can increase the amplitude of spinal motion, withstand pressure, buffer vibration, and protect the brain and spinal cord. It is also the main reason why height changes, but the regulatory mechanism is still unclear, and this study mainly explored the role of miR-874-3p in intervertebral disc degeneration (IDD).

Methods

The mechanism and perform correlation analysis of miR-874-3p and the pathological degree and prognosis of patients with IDD. miR-874-3p is involved in the progression of several diseases, such as cell differentiation, proliferation, apoptosis and extracellular matrix degradation, overexpressing cell line GV369-miR-874-3p-NP was obtained by infected nucleus pulposus (NP) cells, and the empty vector GV369-NP group was set with the blank group. The expression of the tag protein (green fluorescent protein, GFP) was visualized by fluorescence microscopy, followed by real-time polymerase chain reaction (PCR) method to detect miR-874-3p expression, apoptosis by flow cytometry, luciferase reporter analysis for verifying the targeting relationship between miR-874-3p, caspase-3, B-cell lymphoma-2 (Bcl-2) and Bax in cells, and examined the changes in cellular mitochondrial membrane potential using kits.

Results

The expression of miR-874-3p was significantly reduced in IDD patients, and was negatively correlated with matrix metallopeptidase 2 (MMP2) and downregulated matrix metallopeptidase 3 (MMP3) in the NP cells. In addition, western blot revealed that overexpression of miR-874-3p increased the aggregation protein level in the NP cells.

Conclusions

miR-874-3p can inhibit the cell death of IDD, and not only participate in caspase-3 and Fas-associating protein with a novel death domain (FADD)-mediated apoptosis through targeted regulation of exogenous MMP2/MMP3 pathway, but also play a role in cell apoptosis through mitochondrial pathway.

SUBMITTER: Yang X

PROVIDER: S-EPMC8908150 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Publications

Mechanism of protective role of miR-874-3p in intervertebral disc degeneration.

Yang Xuefang X

Annals of translational medicine 20220201 4

<h4>Background</h4>The intervertebral disc can increase the amplitude of spinal motion, withstand pressure, buffer vibration, and protect the brain and spinal cord. It is also the main reason why height changes, but the regulatory mechanism is still unclear, and this study mainly explored the role of miR-874-3p in intervertebral disc degeneration (IDD).<h4>Methods</h4>The mechanism and perform correlation analysis of miR-874-3p and the pathological degree and prognosis of patients with IDD. miR- ...[more]

PMID: 35280407

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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

Mechanism of protective role of miR-874-3p in intervertebral disc degeneration.

Background

Methods

Results

Conclusions

Publications

Mechanism of protective role of miR-874-3p in intervertebral disc degeneration.

Similar Datasets

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