溃疡性结肠炎中microRNA-206对炎症的调控作用及其机制研究

We found miR-206 was upregulated in active ulcerative colitis (UC) tissue in pevious study, and activation of adenosine A3 receptor (A3AR) has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1β expression in colonic epithelial cells and in mice with DSS colitis. Bioinformatics has shown that the 3’-UTR of A3AR mRNA contains a putative binding site for miR-206. So what is the role and mechanism of miR-206 in UC? Though A3AR or not? In this study, the relationship between the miR-206 and A3AR expression, and their localization in colonic epithelial cells is analyzed by qRT-PCR and other methods. The role of miR-206 in colonic inflammation and the effect of miR-206 on A3AR expression are assessed by cell transfection, dual-luciferase reporter assay, gain-of-function and loss-of-function studies and mouse model of DSS colitis. Understanding of the function of miRNA and A3AR and their relationship with UC would help to further elucidate the pathogenesis of UC. Our study will provide scientific basis to establish miRNA as a new target for UC diagnosis and treatment.

我们前期研究发现活动期溃疡性结肠炎（UC）组织miR-206表达上调，并发现结肠上皮细胞腺苷A3受体（A3AR）的激活可抑制人结肠上皮细胞及小鼠DSS结肠炎组织NF-κB的活化及下游炎症因子IL-8 和 IL-1β的表达，具有抗炎作用。通过预测软件发现miR-206与A3AR mRNA 3′-UTR有潜在结合位点。那么miR-206在UC中的作用及其机制如何？是否通过A3AR发挥作用？本研究采用qRT-PCR等方法检测UC组织中miR-206及A3AR的表达，了解两者的相关性及其在结肠黏膜上皮细胞共定位情况，并通过细胞转染、双荧光素酶报告基因、功能获得性及缺失性实验、小鼠DSS结肠炎模型等方法研究miR-206在UC中的作用及对A3AR的调控作用。通过了解miR-206和A3AR的功能及其与UC的关系，有助于进一步阐明UC的发病机理，为确立miRNA作为UC诊断及治疗新靶点提供科学依据。

越来越多研究显示miRNAs在溃疡性结肠炎（UC）中表达异常，在UC的发生发展、诊断、治疗中发挥作用。文献报道miR-206在UC组织表达上调，但其作用及机制尚不清楚。本研究采用qRT-PCR等方法检测UC组织中miR-206及腺苷A3受体（A3AR）的表达，了解两者的相关性，并通过细胞转染、双荧光素酶报告基因、功能获得性及缺失性实验、小鼠DSS结肠炎模型等方法探讨miR-206在UC中的作用及对A3AR的调控。研究发现，UC组miR-206表达上调，A3AR表达下调，miR-206与A3AR蛋白表达呈显著的负相关关系。免疫荧光检测提示A3AR在结肠上皮细胞胞膜表达。miR-206 mimics转染后的HT-29细胞中A3AR表达水平明显下调；反之，miR-206 inhibitor转染后，A3AR表达明显上调。双荧光素酶报告基因检测证实A3AR的3′-UTR有miR-206结合位点。将 miR-206 mimics转染入HT-29细胞后，再用 TNF-α刺激 HT-29细胞，检测胞核 NF-κB p65表达升高，p-IκB-α、IKKα、p-IKKα、IL-8和IL-1β表达水平升高。而转染miR-206 inhibitor 后，胞核NF-κB p65表达降低，p-IκB-α、IKKα、p-IKKα、IL-8和IL-1β表达水平下降。进一步实验显示，将miR-206 inhibitor和A3AR-siRNA 共转染入 HT-29细胞，发现A3AR-siRNA 能部分逆转miR-206 inhibitor的抗炎效应。在DSS诱导的小鼠结肠炎中，给予miR-206 agomir后加重结肠炎的严重程度（体重明显下降，疾病活动指数DAI明显升高；结肠明显缩短，MPO活性增强），而给予miR-206 antagomir后，上述指标均有改善。同样地，给予miR-206 agomir后，DSS结肠炎小鼠的结肠组织A3AR表达下调，胞核 NF-κB p65、TNF-α、IL-8 和 IL-1β表达水平均上调；给予miR-206 antagomir后，A3AR表达上调，胞核 NF-κB p65、TNF-α、IL-8 和 IL-1β表达水平均降低。综上所述，我们的研究结果显示，miR-206通过抑制靶基因A3AR表达，促进NF-κB信号通路的活化，从而在UC的发生发展中发挥促炎作用。