Background & Aims

Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets and functional consequences are unknown.

Methods

microRNA-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative polymerase chain reaction in colonic IECs. Functional characterization of activin receptor-like kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan–Meier estimator.

Results

ALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3’-untranslated region reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in a 5-ethynyl-2-deoxyuridine proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling increased colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared with healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a transepithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and was associated with a higher risk of endoscopic relapse in CD patients.

Conclusions

Decreased colonic ALK1 disrupted colonic IEC barrier integrity and was associated with poor clinical outcomes in CD patients.

中文翻译：

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结肠激活素受体样激酶 1 的减少会破坏克罗恩病患者的上皮屏障完整性。

背景与目标

肠上皮细胞 (IEC) 屏障功能障碍对克罗恩病 (CD) 的发展至关重要。然而，该机制尚未得到充分研究。我们最近报道了 CD 患者结肠 IEC 中 microRNA-31-5p (miR-31-5p) 表达增加，但下游靶点和功能后果尚不清楚。

方法

通过对来自结肠粘膜的 RNA 和小 RNA 测序数据的综合分析鉴定 microRNA-31-5p 靶基因，并通过结肠 IEC 中的定量聚合酶链反应确认。IEC 中激活素受体样激酶 1（ACVRL1 或 ALK1）的功能表征是使用二维培养的人原代结肠 IEC 离体进行的。通过多变量回归分析和 Kaplan-Meier 估计量评估 CD 中结肠 ALK1 信号改变对手术和内镜下复发风险的影响。

结果

ALK1在 CD 患者的结肠 IEC 中被鉴定为 miR-31-5p 的靶标，并使用 3'-非翻译区报告基因测定法证实。在 5-乙炔基-2-脱氧尿苷增殖试验中，ALK1 的激活限制了结肠 IEC 的增殖，并下调了干性相关基因的表达。激活的 ALK1 信号增加了结肠 IEC 向结肠细胞的分化。与健康对照组相比，下调的 ALK1 信号传导与 CD 患者结肠 IEC 中干性增加和结肠细胞特异性标志物表达降低有关。在跨上皮电阻通透性测定中，ALK1 的激活增强了上皮屏障的完整性。下结肠ALK1表达被确定为手术的独立危险因素，并且与 CD 患者内镜下复发的风险较高有关。

结论

结肠 ALK1 的降低破坏了结肠 IEC 屏障的完整性，并与 CD 患者的不良临床结果相关。