咳嗽变异性哮喘患儿外周血miR-138及RUNX3对Th1/Th2平衡的调节作用

Regulatory effects of miR-138 and RUNX3 on Th1/Th2 balance in peripheral blood of children with cough variant asthma

摘要:

目的 检测咳嗽变异性哮喘（cough variant asthma，CVA）患儿外周血中微小RNA-138（microRNA-138，miR-138）、Runt相关转录因子3（Runt-related transcription factor 3，RUNX3）的表达水平及其对Th1/Th2平衡的调节作用。 方法 选取CVA患儿65例纳入CVA组，健康儿童30例纳入健康对照组，采集两组儿童外周静脉血，分离培养CD4⁺T细胞。ELISA法检测CD4⁺T细胞分泌的干扰素-γ（interferon-γ，IFN-γ）、白细胞介素（interleukin，IL）-2、IL-4、IL-5和IL-13的水平；流式细胞仪检测Th1、Th2细胞比例；实时荧光定量PCR检测CD4⁺T细胞中RUNX3 mRNA水平和外周血中miR-138水平；Western blot检测CD4⁺T细胞中RUNX3蛋白表达水平。双荧光素酶报告基因实验检测miR-138和RUNX3的靶向作用；RUNX3-mimic质粒转染至CD4⁺T细胞中，检测增加RUNX3的表达水平对IFN-γ、IL-2、IL-4、IL-5、IL-13水平和Th1、Th2细胞比例的影响。 结果 CVA组与健康对照组相比，CD4⁺T细胞分泌IFN-γ、IL-2水平降低，IL-4、IL-5和IL-13水平增加，Th1细胞比例和Th1/Th2比值降低，Th2细胞比例增加（P<0.05）。CVA组外周血中CD4⁺T细胞RUNX3 mRNA及其蛋白相对表达水平低于健康对照组（P<0.001），miR-138的相对表达水平高于健康对照组（P<0.001）。miR-138可靶向调节RUNX3的表达。CD4⁺T细胞中RUNX3的表达增加后，IFN-γ和IL-2水平升高，IL-4、IL-5和IL-13水平降低，Th1细胞比例和Th1/Th2比值增加，Th2细胞比例降低（P<0.05）。 结论 miR-138通过靶向RUNX3调节CVA患儿Th1/Th2平衡，为CVA的治疗提供了新方向。

Abstract:

Objective To study the expression levels of microRNA-138 (miR-138) and Runt-related transcription factor 3 (RUNX3) in peripheral blood of children with cough variant asthma (CVA) and their regulatory effects on Th1/Th2 balance. Methods Sixty-five children with CVA (CVA group) and 30 healthy children (control group) were enrolled. Peripheral venous blood samples were collected for both groups, and CD4+ T cells were isolated and cultured. Enzyme-linked immunosorbent assay was used to measure the levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-5, and IL-13 that were secreted by CD4+ T cells. Flow cytometry was used to determine the percentages of Th1 and Th2 cells. Quantitative real-time PCR was used to measure the level of RUNX3 mRNA in CD4+ T cells and the level of miR-138 in peripheral blood. Western blot was used to determine the protein expression of RUNX3 in CD4+ T cells. The dual-luciferase reporter assay was used to determine the targeting effects of miR-138 and RUNX3. The RUNX3-mimic plasmid was transfected into CD4+ T cells, and the effects on the levels of IFN-γ, IL-2, IL-4, IL-5, and IL-13 and the percentages of Th1 and Th2 cells were measured. Results Compared with the control group, the CVA group showed significantly decreased levels of IFN-γ and IL-2 from CD4+ T cells, significantly increased levels of IL-4, IL-5, and IL-13 from CD4+ T cells, significantly decreased Th1 cell percentage and Th1/Th2 ratio, and a significantly increased Th2 cell percentage (P<0.05). The CVA group showed significantly lower relative expression levels of RUNX3 mRNA and protein in CD4+ T cells in peripheral blood than the control group (P<0.001). The relative expression level of miR-138 was significantly higher in the CVA group than in the control group (P<0.001). MiR-138 could target the expression of RUNX3. Upregulating the expression of RUNX3 in CD4+ T cells induced significantly increased levels of IFN-γ and IL-2, significantly decreased levels of IL-4, IL-5, and IL-13, significantly increased Th1 cell percentage and Th1/Th2 ratio, and a significantly decreased Th2 cell percentage (P<0.05). Conclusions MiR-138 regulates Th1/Th2 balance by targeting RUNX3 in children with CVA, providing a new direction for the treatment of CVA.