微小RNA-455-3p调控Toll样受体4对哮喘大鼠气道平滑肌细胞的影响

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

  • 摘要:
    目的 探讨微小RNA-455-3p(miR-455-3p)对哮喘大鼠气道平滑肌细胞(ASMC)增殖、凋亡及炎症因子分泌功能的影响及可能机制。
    方法 建立卵清蛋白(OVA)诱导的哮喘大鼠模型(模型组),同时设立正常对照组(正常大鼠,等量生理盐水),采用实时荧光定量聚合酶链反应(qRT-PCR)检测2组大鼠肺组织miR-455-3p表达水平; 采用酶联免疫吸附试验(ELISA)检测肺组织和血清中炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平。分离大鼠ASMC,采用双荧光素酶报告基因实验验证miR-455-3p与Toll样受体4(TLR4)的靶向关系; 使用TNF-α刺激哮喘大鼠ASMC产生炎症反应。通过Lip2000转染法转染miR-455-3p NC(miR-455-3p NC组)、miR-455-3p mimic(miR-455-3p mimic组)和共转染miR-455-3p mimic与pcDNA(miR-455-3p mimic+pcDNA组)、miR-455-3p mimic与pcDNA-TLR4(miR-455-3p mimic+pcDNA-TLR4组)于哮喘大鼠炎症ASMC, 另设正常对照组(正常大鼠ASMC)和模型组(哮喘大鼠炎症ASMC), 采用四甲基偶氮唑蓝(MTT)法检测细胞增殖情况,采用原位末端标记法(TUNEL)检测细胞凋亡情况,采用ELISA检测细胞上清液中TNF-α、IL-6水平,采用qRT-PCR和免疫印迹法(WB)分别检测miR-455-3p、TLR4 mRNA及TLR4蛋白表达水平。
    结果 模型组大鼠肺组织中miR-455-3p表达水平低于正常对照组,且肺组织与血清中TNF-α、IL-6表达水平均高于正常对照组,差异有统计学意义(P < 0.05)。双荧光素酶报告基因实验结果显示, miR-455-3p可直接靶向抑制TLR4基因表达。与正常对照组相比,模型组大鼠ASMC中miR-455-3p表达水平、ASMC增殖活性降低, TLR4 mRNA及TLR4蛋白表达水平、凋亡率和ASMC上清液TNF-α、IL-6水平升高,差异有统计学意义(P < 0.05); 与miR-455-3p NC组相比, miR-455-3p mimic组ASMC中miR-455-3p表达水平、ASMC增殖活性升高, TLR4 mRNA及TLR4蛋白表达水平、凋亡率和ASMC上清液TNF-α、IL-6水平降低,差异有统计学意义(P < 0.05); 与miR-455-3p mimic+pcDNA组比较, miR-455-3p mimic+pcDNA-TLR4组ASMC中miR-455-3p表达水平、ASMC增殖活性降低, TLR4 mRNA及TLR4蛋白表达水平、凋亡率和ASMC上清液TNF-α、IL-6水平升高,差异有统计学意义(P < 0.05)。
    结论 miR-455-3p可通过靶向抑制TLR4表达,减轻哮喘大鼠ASMC炎症反应,调节其增殖与凋亡平衡。

     

    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.

     

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