Abstract

Background and Aims

Mucosal healing is important in Crohn’s disease therapies. Epithelial homeostasis becomes dysregulated in Crohn’s, with increased permeability, inflammation, and diarrhoea. MicroRNAs are small non-coding RNAs that regulate gene expression and show changes in inflammatory bowel disease. Tumour necrosis factor alpha [TNFα] inhibitor protein 3 is raised in Crohn’s and regulates TNFα-mediated activation of NFκB. We investigated TNFα regulation by microRNA in Crohn’s disease [CD], and studied effects on epithelial permeability and inflammation.

Methods

Colonic epithelium from CD and healthy donor biopsies was isolated using laser capture microdissection, and microRNA was quantified. Tumour necrosis factor alpha inhibitor protein 3 was characterised immunohistochemically on serial sections. Expression effect of microRNA was confirmed with luciferase reporter assays. Functional barrier permeability studies and innate cytokine release were investigated with cell and explant culture studies.

Results

MicroRNA23a levels significantly increased in colonic Crohn’s epithelium compared with healthy epithelium. Luciferase reporter assays in transfected epithelial cells confirmed that microRNA23a repressed expression via the 3’ untranslated region of tumour necrosis factor alpha inhibitor protein 3 mRNA, coinciding with increased NFκB-mediated transcription. Immunohistochemical staining of TNFAIP3 protein in colonic biopsies was reduced or absent in adjacent Crohn’s sections, correlating inversely with microRNA23a levels and encompassing some intercohort variation. Overexpression of microRNA23a increased epithelial barrier permeability in a colonic epithelial model and increased inflammatory cytokine release in cultured explant biopsies, mimicking Crohn’s disease characteristics.

Conclusions

MicroRNA23a overexpression in colonic Crohn’s epithelium represses tumour necrosis factor alpha inhibitor protein 3, enhancing sensitivity to TNFα, with increased intestinal permeability and cytokine release.

中文翻译：

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MicroRNA23a在克罗恩病中的过量表达靶向肿瘤坏死因子α抑制剂蛋白3，增加对TNF的敏感性并修饰上皮屏障

抽象的

背景和目标

粘膜愈合在克罗恩病治疗中很重要。克罗恩氏病中上皮稳态失调，通透性，炎症和腹泻增加。MicroRNA是调节基因表达并在炎症性肠病中显示变化的小型非编码RNA。肿瘤坏死因子α[TNFα]抑制剂蛋白3在克罗恩病中产生，并调节TNFα介导的NFκB活化。我们研究了microRNA在克罗恩病[CD]中的TNFα调节，并研究了对上皮通透性和炎症的影响。

方法

使用激光捕获显微切割技术从CD和健康的供体活组织检查中分离出结肠上皮，并对microRNA进行定量。在连续切片上免疫组织化学表征肿瘤坏死因子α抑制剂蛋白3。荧光素酶报告基因检测证实了microRNA的表达效果。通过细胞和外植体培养研究对功能性屏障通透性研究和先天细胞因子释放进行了研究。

结果

与健康上皮相比，结肠克罗恩上皮中的MicroRNA23a水平显着增加。在转染的上皮细胞中的荧光素酶报告基因检测证实，microRNA23a通过肿瘤坏死因子α抑制剂蛋白3 mRNA的3'非翻译区抑制表达，与增加的NFκB介导的转录相符。结肠活检组织中TNFAIP3蛋白的免疫组织化学染色在邻近的克罗恩氏切片中减少或消失，与microRNA23a水平成反比，并包含一些队列间变异。microRNA23a的过表达在结肠上皮模型中增加了上皮屏障的通透性，在培养的外植体活检中增加了炎性细胞因子的释放，模仿了克罗恩病的特征。

结论

结肠克罗恩上皮中的MicroRNA23a过表达抑制肿瘤坏死因子α抑制剂蛋白3，增强了对TNFα的敏感性，并增加了肠道通透性和细胞因子释放。