Division of Gastroenterology & Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
Jesse Brown VA Medical Center, Chicago, Illinois.
Am J Physiol Cell Physiol. 2022 Dec 1;323(6):C1720-C1727. doi: 10.1152/ajpcell.00294.2022. Epub 2022 Oct 3.
Na/H exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.
钠/氢交换器-3（NHE-3）是肠道中涉及向量 Na 吸收的主要顶膜转运蛋白。NHE-3 表达和/或功能的失调与肠道炎症和感染相关的腹泻的病理生理学有关。因此，了解调节 NHE-3 表达的机制至关重要。微小 RNA（miRNA）是高度保守的小 RNA，可以在转录后水平调节基因表达。然而，迄今为止，miRNA 对 NHE-3 表达的调节知之甚少。因此，目前的研究旨在研究可以调节肠上皮细胞中 NHE-3 表达的潜在 miRNA 候选物。使用不同的算法进行计算机分析，预测了几种靶向 NHE-3 的 miRNA。选择具有最高上下文和靶评分的 miRNA，即 miR-326、miR-744-5p 和 miR-330-5p，用于当前研究。人 NHE-3 基因 3'非翻译区 [3'UTR；160 个碱基对 (bp)] 被克隆到 pmirGLO 载体上游的荧光素酶报告基因中，并将 miR-326、miR-744-5p 和 miR-330-5p 的模拟物瞬时转染到 Caco-2、HT-29 和 SK-CO15 细胞中。NHE-3 3'UTR 与 miR-326 和 -miR-330-5p 模拟物的共转染导致相对荧光素酶活性显著降低。miR-326 和 -330-5p 模拟物转染 SK-CO15 细胞显著降低了 NHE-3 蛋白表达，而 NHE-3 信使核糖核酸（mRNA）水平没有变化。我们的研究结果表明，miR-326 和 -330-5p 通过翻译抑制对 NHE-3 进行转录后调节的新机制。我们推测，miR-326 和 -330-5p 依赖的途径可能参与调节生理和病理生理条件下的 NHE-3 表达。
