Antibody information used in this study.

Irritable bowel syndrome with predominant diarrhea (IBS-D) is characterized by increased intestinal permeability. Previous studies have shown that the microRNA-29 gene is involved in the regulation of intestinal permeability in patients with IBS-D. NF-κB was proved to play a key role in inflammatory response of intestine and resultant disruption of tight junction integrity, whose activity could be inhibited by TNF Receptor-Associated Factor 3 (TRAF3). However, the exact mechanism that induces increased intestinal permeability in IBS-D patients has not been clarified. In this study, we found that microRNA-29b‑3p (miR-29b-3p) was significantly upregulated, while TRAF3 was decreased and the NF-κB-MLCK pathway was activated within the colonic tissue of IBS-D patients. Subsequently, we confirmed the targeting relationship between miR-29b-3p and TRAF3 through a double-luciferase reporter assay. Lentivirus transfection of NCM460 cells with miR-29b-3p-overexpressing and -silencing vectors demonstrated that the expression of TRAF3 was negatively correlated with the level of miR-29b-3p. The NF-κB/MLCK pathway was activated in the miR-29b-3p-overexpressing group and inhibited to some extent in the miR-29b-3p-silencing group. Results in WT and miR-29 knockout mice showed that miR-29b-3p levels were increased, TRAF3 levels were decreased, and the NF-κB/MLCK signaling was activated in the WT IBS-D group as compared with the WT control group. The protein levels of TRAF3 and TJs in the miR-29b-/- IBS-D group were partially recovered and NF-κB/MLCK pathway indicators were, to a certain extent, decreased as compared with the WT IBS-D group. These results suggested that miR-29b-3p deletion enhances the TRAF3 level in IBS-D mice and alleviates the high intestinal permeability. In brief, through the analysis of intestinal tissue samples from IBS-D patients and miR-29b-/- IBS-D mice, we showed that miR-29b-3p is involved in the pathogenesis of intestinal hyperpermeability in IBS-D via targeting TRAF3 to regulate the NF-κB-MLCK signaling pathway.

以腹泻为主要表型的肠易激综合征（Irritable Bowel Syndrome with Predominant Diarrhea, IBS-D）以肠道通透性升高为核心特征。既往研究证实，microRNA-29基因参与调控IBS-D患者的肠道通透性。核因子κB（Nuclear Factor-kappa B, NF-κB）已被证明在肠道炎症反应及由此引发的紧密连接完整性破坏过程中发挥关键作用，其活性可被肿瘤坏死因子受体相关因子3（Tumor Necrosis Factor Receptor-Associated Factor 3, TRAF3）抑制。然而，目前尚未阐明诱导IBS-D患者肠道通透性升高的确切分子机制。本研究发现，IBS-D患者结肠组织中microRNA-29b-3p（miR-29b-3p）表达显著上调，而TRAF3表达降低，且NF-κB-肌球蛋白轻链激酶（Myosin Light Chain Kinase, MLCK）通路被激活。随后通过双荧光素酶报告基因实验，验证了miR-29b-3p与TRAF3的靶向结合关系。利用过表达及沉默miR-29b-3p的慢病毒载体转染NCM460细胞的实验证实，TRAF3的表达水平与miR-29b-3p含量呈负相关。在miR-29b-3p过表达组中，NF-κB/MLCK通路被激活；而在miR-29b-3p沉默组中，该通路受到一定程度的抑制。野生型（Wild Type, WT）及miR-29基因敲除小鼠的实验结果显示，与野生型对照组相比，野生型IBS-D组小鼠的miR-29b-3p水平升高、TRAF3表达降低，且NF-κB/MLCK信号通路被激活。与野生型IBS-D组相比，miR-29b基因敲除（miR-29b-/-）IBS-D组小鼠的TRAF3及紧密连接（Tight Junctions, TJs）蛋白水平部分恢复，NF-κB/MLCK通路相关指标也出现一定程度的下调。上述结果表明，敲除miR-29b-3p可提升IBS-D小鼠体内TRAF3的表达水平，并缓解肠道高通透性状态。简言之，通过分析IBS-D患者结肠组织样本及miR-29b基因敲除（miR-29b-/-）IBS-D小鼠模型，本研究证实miR-29b-3p通过靶向TRAF3调控NF-κB-MLCK信号通路，参与IBS-D肠道高通透性的发病过程。