Up-Regulation of microRNA-126 May Contribute to Pathogenesis of Ulcerative Colitis via Regulating NF-kappaB Inhibitor IκBα

BackgroundMicroRNAs (miRNAs) are important post-transcriptional regulators. Altered expression of miRNAs has recently demonstrated association with human ulcerative colitis (UC). In this study, we attempted to elucidate the roles of miR-126 in the pathogenesis of UC.
MethodsExpression of miR-126, miR-21, miR-375 and the potential targets NF-κB inhibitor alpha (IκBα, IKBA or NFKBIA), Polo-like kinase 2 (PLK2) and v-Crk sarcoma virus CT10 oncogene homolog (CRK) were assessed in 52 colonic biopsies from patients with active UC, inactive UC, irritable bowel syndrome (IBS) and from healthy subjects by quantitative RT-PCR and immunofluorescence analyses. Regulation of gene expression by miR-126 was assessed using luciferase reporter construct assays and specific miRNA mimic transfection.
ResultsWe found that the expression of miR-126 and miR-21 were significantly increased in active UC group compared to the inactive UC, IBS and healthy control groups (P<0.05). In contrast, the expression of IKBA mRNA and protein was remarkably decreased in the active UC group compared with the other three groups (P<0.05). The expression of miR-126 and IKBA mRNA were inversely correlated in active UC patients (P<0.05). However the expression of miR-375, PLK2 and CRK showed no difference between each group. Furthermore, we demonstrate that endogenous miR-126 and exogenous miR-126 mimic can inhibit IκBα expression. Finally, mutating the miR-126 binding site of the IKBA 3′-UTR reporter construct restored reporter gene expression.
ConclusionmiR-126 may play roles in UC inflammatory activity by down-regulating the expression of IKBA, an important inhibitor of NF-κB signaling pathway.

研究背景
微小RNA（MicroRNAs, miRNAs）是一类重要的转录后调控因子。近期研究证实，miRNA表达异常与人类溃疡性结肠炎（ulcerative colitis, UC）密切相关。本研究旨在阐明miR-126在溃疡性结肠炎发病机制中的作用。

研究方法
本研究通过定量实时逆转录聚合酶链反应（quantitative RT-PCR, qRT-PCR）与免疫荧光分析，对52份结肠活检组织中相关分子的表达水平进行检测。这些活检组织分别取自活动性UC、非活动性UC、肠易激综合征（irritable bowel syndrome, IBS）患者及健康受试者，涉及的分子包括miR-126、miR-21、miR-375，以及潜在靶标核因子κB抑制蛋白α（NF-κB inhibitor alpha, IκBα、IKBA或NFKBIA）、polo样激酶2（Polo-like kinase 2, PLK2）和v-Crk肉瘤病毒CT10癌基因同源物（v-Crk sarcoma virus CT10 oncogene homolog, CRK）。本研究采用荧光素酶报告基因构建体实验（luciferase reporter construct assays）与特异性miRNA模拟物转染实验，评估miR-126对基因表达的调控作用。

研究结果
与非活动性UC、IBS及健康对照组相比，活动性UC组中miR-126与miR-21的表达水平显著升高（P<0.05）。与之相反，活动性UC组中IKBA的mRNA及蛋白表达水平较其余三组显著降低（P<0.05）。活动性UC患者体内miR-126与IKBA mRNA的表达水平呈显著负相关（P<0.05）。而miR-375、PLK2及CRK的表达在各组间均无显著差异。此外，本研究证实内源性miR-126与外源性miR-126模拟物均可抑制IκBα的表达。对IKBA 3′非翻译区（3′-UTR）报告基因构建体的miR-126结合位点进行突变后，报告基因的表达得以恢复。

研究结论
miR-126可通过下调核因子κB（NF-κB）信号通路的关键抑制因子IKBA的表达，参与溃疡性结肠炎的炎症活性调控。