The changes of microRNA in nasal mucosa after the specific immunotherapy for allergic rhinitis in mice
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摘要: 目的: 研究变应性鼻炎(AR)特异性免疫治疗(SIT)前后鼻黏膜microRNA表达的变化。方法: 6~8周龄雌性BALB/c小鼠随机分为空白组、模型组和治疗组。采用卵清蛋白(OVA)腹腔注射及滴鼻法建立AR小鼠模型,以腹股沟皮下注射法进行SIT,以叠加法进行行为学评分,计算鼻黏膜中嗜酸粒细胞个数,ELISA检测血清中特异性OVA-slgE的表达及鼻腔灌洗液中IFN-γ与IL-4的表达。3组小鼠分别用microRNA基因芯片进行初步筛查,筛查治疗组与模型组相比Fold change ≥ 2.0且P<0.05的microRNA,而后用GeneSpring12.5软件进行靶基因预测并与免疫反应和炎症相关通路中的mRNA取交集。用MEV-4-6-0软件做聚类分析图,Cytoscape_v2.8.2做靶基因网络图。结果: AR小鼠模型及SIT成功。治疗组与模型组相比有9个microRNA表达下调,6个microRNA表达上调。聚类分析可明显看出治疗组和模型组分别聚在2个分支。这15个microRNA的靶基因有5753个,取交集后得到451个与SIT相关性更大的靶基因。从网络图看出,一个microRNA可以调控多个靶基因,一个基因也可以受多个microRNA的调控,它们的相互作用可能参与了SIT的过程。结论: 鼻黏膜microRNA的表达在AR进行SIT前后发生了变化,microRNA可能参与了AR的SIT过程;通过生物信息学方法缩小microRNA靶基因的查找范围,有利于研究AR在SIT后变化的microRNA对其相关靶基因表达的影响,为AR的发病和SIT机制的认识提供了依据。Abstract: Objective: To explore the changes of microRNAs in nasal mucosa after the specific immunotherapy(SIT) for allergic rhinitis(AR) in mice.Method: Female BALB/c mice, 6-8 weeks of age, were randomly divided into control group, model group and treatment group. AR model were established by intraperitoneal injection and intranasal challenge of ovalbumin and SIT was performed by inguinal subcutaneous injections. AR symptom scores were documented. The eosinophils (EOS) in the nasal mucosa were measured. Ovalbumin-specific IgE(OVA-sIgE) in the serum and expression of interferon-γ and interleukin-4 in the nasal lavage were measured by enzyme-linked immunosorbent assay meanwhile the ratio of interferon-γ and interleukin-4 was calculated. The microRNAs in the nasal mucosa were preliminary screened by microRNA gene microarray. Comparing with model group, the Fold changes of microRNA of the treatment group were ≥ 2.0 and the P<0.05. MicroRNA target genes were predicted with GeneSpring12.5 software. We took the intersection between genes in the signal pathway which associated with immune response,inflammation and target genes. The MEV-4-6-0 and Cytoscape_v2.8.2. software was applied to perform the cluster analysis and target gene regulatory networks maps.Result: The model of AR in mice and its SIT were successful. Comparing with the model group, the Fold changes of 15 microRNAs, of which 9 microRNAs were up-regulated and 6 microRNAs were down-regulated, were ≥ 2.0 in treatment group (P<0.05). Cluste analysis showed clearly that microRNAs in the treatment group and model group respectively aggregated in two branches. The 15 microRNAs had 5302 target genes, of which, 451 genes were related more with SIT by the intersection. One microRNA can regulate many target genes, and one gene can also be affected by many microRNAs. Their synergistic effects may be involved in the mechanism of SIT.Conclusion: The expressions of microRNAs are changed in nasal mucosa after SIT for AR in mice and we can speculate that microRNAs are involved in the process of SIT for AR. Bioinformatics methods can diminish the scope of target genes of microRNAs, which will help us studying the effect of changed microRNA on its relative target genes after SIT, and make us better understanding the mechanism of the disease and its SIT.
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Key words:
- rhinitis /
- allergic /
- specific immunotherapy /
- gene chips /
- cluster analysis /
- gene regulatory networks
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