Effects of microRNA-455-3p on airway smooth muscle cells in asthmatic rats by regulating toll-like receptor 4

XUE Mei; LIAO Yi; LU Wei

doi:10.7619/jcmp.20221809

Journal of Clinical Medicine in Practice > 2022 > 26(22): 25-32. > DOI: 10.7619/jcmp.20221809

XUE Mei, LIAO Yi, LU Wei. Effects of microRNA-455-3p on airway smooth muscle cells in asthmatic rats by regulating toll-like receptor 4[J]. Journal of Clinical Medicine in Practice, 2022, 26(22): 25-32. DOI: 10.7619/jcmp.20221809

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Effects of microRNA-455-3p on airway smooth muscle cells in asthmatic rats by regulating toll-like receptor 4

Department of Pediatrics, Chengdu Hospital of Integrated Traditional and Western Medicine, Chengdu, Sichuan, 610000

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Received Date: June 08, 2022
Available Online: December 01, 2022

Graphical Abstract

Abstract

Abstract

Objective
To investigate the effects of microRNA-455-3p (miR-455-3p) on the proliferation, apoptosis and secretion function of inflammatory factors of airway smooth muscle cells (ASMC) in asthmatic rats and its possible mechanism.
Methods
The ovalbumin (OVA)-induced asthmatic rat model (model group) and the normal control group (normal rats with the same amount of normal saline) were established. The expression level of miR-455-3p in lung tissue was detected by real-time fluorescent quantitative PCR (qRT-PCR), the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in lung tissue and serum were detected by Enzyme-linked immunosorbent assay (ELISA). Rat ASMC were isolated, double luciferase reporter gene assay was used to verify the targeting relationship between miR-455-3p and toll like receptor 4 (TLR4). TNF-α was used to stimulate the ASMC of asthmatic rats to produce inflammation. The Lip2000 transfection method was used to transfect miR-455-3p NC (miR-455-3p NC group), miR-455-3p mimic (miR-455-3p mimic group), and co-transfect miR-455-3p mimic and pcDNA (miR-455-3p mimic+pcDNA group), miR-455-3p mimic and pcDNA-TLR4 (miR-455-3p mimic+ pcDNA-TLR4 group) to inflammatory ASMC of asthmatic rats. ASMC of normal rats was set as normal control group, and ASMC with inflammation of asthmatic rats was set as model group. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell proliferation, apoptosis was detected by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), the levels of TNF-α and IL-6 in the supernatant were detected by ELISA, in addition, the expression levels of miR-455-3p, TLR4 mRNAs and TLR4 protein were detected by qRT-PCR and western blot (WB).
Results
Compared with the normal control group, the expression of miR-455-3p in the lung tissue of the model group was significantly reduced, and the levels of TNF-α and IL-6 in the lung tissue and serum were significantly increased (P < 0.05). Double luciferase reporter gene assay showed that miR-455-3p could directly target and inhibit TLR4 gene expression. Compared with the normal control group, the expression level of miR-455-3p in ASMC and the proliferation activity of ASMC reduced significantly, and the expression levels of TLR4 mRNA and TLR4 protein, apoptosis rate and TNF-α and IL-6 levels in ASMC supernatant increased significantly in the model group (P < 0.05); compared with the miR-455-3p NC group, the expression level of miR-455-3p and the proliferation activity of ASMC increased significantly, TLR4 mRNA and TLR4 protein expression levels, apoptosis rate and TNF-α and IL-6 levels in ASMC supernatant were significantly reduced in the miR-455-3p mimic group(P < 0.05); compared with miR-455-3p mimic+pcDNA group, miR-455-3p expression level and ASMC proliferation activity in ASMC reduced significantly, TLR4 mRNA and TLR4 protein expression levels, apoptosis rate and TNF-α and IL-6 levels in ASMC supernatant increased significantly in miR-455-3p mimic+pcDNA-TLR4 group (P < 0.05).
Conclusion
MiR-455-3p can relieve the inflammatory response and regulate the balance of proliferation and apoptosis of ASMC in asthmatic rats by targeted inhibition of the expression of TLR4.
- asthma,
- rats,
- airway smooth muscle cells,
- microRNA-455-3p,
- toll-like receptor 4,
- proliferation,
- apoptosis,
- inflammation

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Effects of microRNA-455-3p on airway smooth muscle cells in asthmatic rats by regulating toll-like receptor 4

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