MiR-124在克罗恩病中的表达及其靶基因的确定

Expression of miR-124 in active Crohn's disease and validation of aryl hydrocarbon receptor as its target gene

目的:检测活动期克罗恩病(Crohn's disease,CD)患者病变肠道黏膜组织中microRNA-124(miR-124)的表达情况并确定其靶基因.方法:采用定量PCR技术检测活动期CD患者及健康对照者肠道黏膜组织中miR-124的表达情况,使用Targetscan等多种软件预测得出芳香烃受体(aryl hydrocarbon receptor,AHR)是miR-124的靶基因之一,利用荧光素酶报告基因实验验证miR-124和AHR的靶点关系,并通过在Caco-2细胞中过表达或抑制表达miR-124进一步检测AHR的变化.结果:与健康对照者相比,miR-124在活动期CD患者病变肠道黏膜组织中明显升高,其相对表达量为7.19±3.54(P<0.01).荧光素酶报告基因结果提示在Caco-2细胞中miR-124可与AHR的3'端非翻译区特异性结合.在Caco-2细胞中过表达或抑制表达miR-124,可分别使AHR蛋白水平下降或升高,而AHR mRNA水平不受影响.结论:miR-124在活动期CD患者病变肠道黏膜组织中表达明显升高,AHR是其靶点之一.

AIM： To detect the expression of miR-124 in active Crohn＇s disease （CD） and to validate aryl hydrocarbon receptor （AHR） as its target gene.METHODS： Levels of miR-124 in intestinal tissues of active CD patients and healthy controls were assessed by qRT-PCR. AHR was predicted to be a target of miR-124 by bioinformatics analysis. Luciferase reporter assay and Western blot analysis in combination with overexpression or knockdown of miR-124 were performed to experimentally validate if AHR is the genuine target of miR-124.RESULTS： The expression of miR-124 was significantly increased in active CD patients compared with healthy controls （fold change 〉 7, P 〈 0.01）. The luciferase reporter assay confirmed that miR-124 directly combined with the 3’UTR of AHR. In vitro, over-expression of miR-124 led to decreased AHR expression, while knockdown of miR-124 resulted in increased AHR expression, although AHR mRNA expression was not altered.CONCLUSION： Levels of miR-124 are increased in active CD patients and AHR is a target of miR-124.