Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico.
Penn State Health Milton S. Hershey Medical Center, Hershey, Pennsylvania.
Gastroenterology. 2020 Oct;159(4):1375-1389. doi: 10.1053/j.gastro.2020.06.038. Epub 2020 Jun 20.
BACKGROUND & AIMS: Defects in the epithelial tight junction (TJ) barrier contribute to development of intestinal inflammation associated with diseases. Interleukin 1 beta (IL1B) increases intestinal permeability in mice. We investigated microRNAs that are regulated by IL1B and their effects on expression of TJ proteins and intestinal permeability.
We used Targetscan to identify microRNAs that would bind the 3' untranslated region (3'UTR) of occludin mRNA; regions that interacted with microRNAs were predicted using the V-fold server and Assemble2, and 3-dimensional models were created using UCSF Chimera linked with Assemble2. Caco-2 cells were transfected with vectors that express microRNAs, analyzed by immunoblots and real-time polymerase chain reaction (PCR), and grown as monolayers; permeability in response to IL1B was assessed with the marker inulin. Male C57BL/6 mice were given intraperitoneal injections of IL1B and intestinal recycling perfusion was measured; some mice were given dextran sodium sulfate to induce colitis and/or gavage with an antagonist to MIR200C-3p (antagomiR-200C) or the nonspecific antagomiR (control). Intestinal tissues were collected from mice and analyzed by histology and real-time PCR; enterocytes were isolated by laser capture microdissection. We also analyzed colon tissues and organoids from patients with and without ulcerative colitis.
Incubation of Caco-2 monolayers with IL1B increased TJ permeability and reduced levels of occludin protein and mRNA without affecting the expression of other transmembrane TJ proteins. Targetscan identified MIR122, MIR200B-3p, and MIR200C-3p, as miRNAs that might bind to the occludin 3'UTR. MIR200C-3p was rapidly increased in Caco-2 cells incubated with IL1B; the antagomiR-200c prevented the IL1B-induced decrease in occludin mRNA and protein and reduced TJ permeability. Administration of IL1B to mice increased small intestinal TJ permeability, compared with mice given vehicle; enterocytes isolated from mice given IL1B had increased expression of MIR200C-3p and decreased levels of occludin messenger RNA (mRNA) and protein. Intestinal tissues from mice with colitis had increased levels of IL1B mRNA and MIR200C-3p and decreased levels of occludin mRNA; gavage of mice with antagomiR-200C reduced levels of MIR200C-3p and prevented the decrease in occludin mRNA and the increase in colonic permeability. Colon tissues and organoids from patients with ulcerative colitis had increased levels of IL1B mRNA and MIR200C-3p compared with healthy controls. Using 3-dimensional molecular modeling and mutational analyses, we identified the nucleotide bases in the occluding mRNA 3'UTR that interact with MIR200C-3p.
Intestine tissues from patients with ulcerative colitis and mice with colitis have increased levels of IL1B mRNA and MIR200C-3p, which reduces expression of occludin by enterocytes and thereby increases TJ permeability. Three-dimensional modeling of the interaction between MIR200C-3p and the occludin mRNA 3'UTR identified sites of interaction. The antagomiR-200C prevents the decrease in occludin in enterocytes and intestine tissues of mice with colitis, maintaining the TJ barrier.
上皮紧密连接（TJ）屏障的缺陷导致与疾病相关的肠道炎症的发展。白细胞介素 1β（IL1B）增加了小鼠的肠道通透性。我们研究了受 IL1B 调控的 microRNAs 及其对 TJ 蛋白表达和肠道通透性的影响。
我们使用 Targetscan 来识别与 occludin mRNA 的 3'非翻译区（3'UTR）结合的 microRNAs；使用 V-fold 服务器和 Assemble2 预测相互作用的区域，并使用 UCSF Chimera 与 Assemble2 连接创建 3 维模型。用表达 microRNAs 的载体转染 Caco-2 细胞，通过免疫印迹和实时聚合酶链反应（PCR）进行分析，并作为单层细胞生长；用标记物乳果糖评估对 IL1B 的通透性。给雄性 C57BL/6 小鼠腹腔内注射 IL1B 并测量肠道再循环灌注；一些小鼠给予葡聚糖硫酸钠以诱导结肠炎和/或用 MIR200C-3p 的拮抗剂（antagomiR-200C）或非特异性拮抗剂（对照）进行灌胃。从小鼠中收集肠组织并进行组织学和实时 PCR 分析；通过激光捕获微切割分离肠细胞。我们还分析了溃疡性结肠炎患者和无溃疡性结肠炎患者的结肠组织和类器官。
与未用 IL1B 孵育的 Caco-2 单层相比，IL1B 孵育增加了 TJ 通透性，降低了 occludin 蛋白和 mRNA 水平，而不影响其他跨膜 TJ 蛋白的表达。Targetscan 鉴定出 MIR122、MIR200B-3p 和 MIR200C-3p 可能与 occludin 3'UTR 结合的 microRNAs。用 IL1B 孵育的 Caco-2 细胞中 MIR200C-3p 迅速增加；antagomiR-200c 可防止 IL1B 诱导的 occludin mRNA 和蛋白减少，并降低 TJ 通透性。与给予载体的小鼠相比，给予 IL1B 的小鼠小肠 TJ 通透性增加；给予 IL1B 的小鼠分离的肠细胞中 MIR200C-3p 表达增加，occludin 信使 RNA（mRNA）和蛋白水平降低。结肠炎小鼠的肠组织中 IL1B mRNA 和 MIR200C-3p 水平升高，occludin mRNA 水平降低；用 antagomiR-200C 灌胃可降低 MIR200C-3p 水平，并防止 occludin mRNA 减少和结肠通透性增加。与健康对照组相比，溃疡性结肠炎患者的结肠组织和类器官中 IL1B mRNA 和 MIR200C-3p 水平升高。使用 3 维分子建模和突变分析，我们确定了 occluding mRNA 3'UTR 中与 MIR200C-3p 相互作用的核苷酸碱基。
溃疡性结肠炎患者和结肠炎小鼠的肠组织中 IL1B mRNA 和 MIR200C-3p 水平升高，降低了肠细胞中 occludin 的表达，从而增加了 TJ 通透性。MIR200C-3p 和 occludin mRNA 3'UTR 之间相互作用的 3 维模型确定了相互作用的部位。antagomiR-200C 可防止结肠炎小鼠的 occludin 在肠细胞和肠组织中的减少，维持 TJ 屏障。
