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Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice
Published on: July 8, 2020
MicroRNA‑195‑5p inhibitor prevents the development of osteoarthritis by targeting REGγ
Yang Shu1, Junpeng Long2, Weixiong Guo3
1Department of Orthopedics, The Sixth Hospital of Wuhan, Affiliated Hospital of Jianghan University, Wuhan, Hubei 430015, P.R. China.
Abstract:
Osteoarthritis (OA) is a common inflammatory joint disease. MicroRNAs (miRNAs/miRs) have been reported to be involved in the pathogenesis of OA; however, the role of miRNAs in OA remains largely unexplained. The purpose of the present study was to investigate the expression and role of miR‑195‑5p in OA, and to further explore the mechanism. The expression level of miR‑195‑5p was measured using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). TargetScan and a luciferase reporter assay were used to reveal the associations between miR‑195‑5p and REGγ (also known as PSME3). To investigate the role of miR‑195‑5p in OA, a cell model of OA was established by treating ATDC5 cells with lipopolysaccharide (LPS). Then an MTT assay was conducted to detect cell proliferation ability, and an Annexin V‑fluorescein isothiocyanate/propidium iodide apoptosis detection kit was used to measure cell apoptosis. In addition, the levels of interleukin (IL)‑1β, IL‑6 and tumor necrosis factor (TNF)‑α were determined using ELISA. Furthermore, gene and protein expression was measured via RT‑qPCR and western blot assay, respectively. The results revealed that miR‑195‑5p was significantly upregulated in the articular cartilage tissues of patients with OA and in LPS stimulated ATDC5 cells. REGγ was a direct target of miR‑195‑5p. The repressed cell proliferation ability and enhanced cell apoptosis of ATDC5 cells induced by LPS were reversed by miR‑195‑5p downregulation. Furthermore, LPS stimulation significantly upregulated the levels of IL‑1β, IL‑6 and TNF‑α, while miR‑195‑5p downregulation markedly reduced the expression of inflammatory factors induced by LPS. The results also revealed that a miR‑195‑5p inhibitor inhibited the LPS induced repression of the Wnt/β‑catenin signaling pathway and activation of nuclear factor (NF)‑κB signaling pathway in ATDC5 cells. Notably, the results of the present study also indicated that all of the effects of the miR‑195‑5p inhibitor on ATDC5 cells were reversed by REGγ silencing. In conclusion, the results indicated that the miR‑195‑5p inhibitor served a protective role in OA by inhibiting chondrocyte apoptosis and inflammatory responses by regulating the Wnt/β‑catenin and NF‑κB signaling pathways.
Insights
MicroRNA-195-5p is upregulated in osteoarthritis (OA) and promotes inflammation and apoptosis. Inhibiting miR-195-5p protects against OA by regulating Wnt/β-catenin and NF-κB pathways.
Area of Science:
- Biomedical research
- Molecular biology
- Cell biology
Background:
- Osteoarthritis (OA) is a prevalent inflammatory joint disease.
- MicroRNAs (miRNAs) are implicated in OA pathogenesis, but their specific roles are unclear.
- miR-195-5p is a potential regulator in OA development.
Purpose of the Study:
- Investigate the expression and function of miR-195-5p in OA.
- Elucidate the underlying molecular mechanisms involving miR-195-5p in OA.
- Determine the therapeutic potential of targeting miR-195-5p in OA.
Main Methods:
- Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for miR-195-5p expression.
- Luciferase reporter assay to confirm REGγ as a direct target of miR-195-5p.
- Cellular assays (MTT, Annexin V/PI) and ELISA to assess cell viability, apoptosis, and inflammatory cytokine levels (IL-1β, IL-6, TNF-α) in lipopolysaccharide (LPS)-induced OA models.
Main Results:
- miR-195-5p was significantly upregulated in OA cartilage and LPS-treated ATDC5 cells.
- miR-195-5p directly targets REGγ.
- Downregulation of miR-195-5p reversed LPS-induced inhibition of cell proliferation, enhanced apoptosis, and elevated inflammatory cytokine levels.
- miR-195-5p inhibition suppressed Wnt/β-catenin pathway repression and NF-κB pathway activation.
- REGγ silencing reversed the protective effects of the miR-195-5p inhibitor.
Conclusions:
- miR-195-5p exacerbates OA by promoting chondrocyte apoptosis and inflammation.
- Targeting miR-195-5p offers a protective role in OA via modulation of Wnt/β-catenin and NF-κB signaling pathways.
- REGγ acts as a downstream mediator of miR-195-5p's effects in OA pathogenesis.
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