在Caco-2肠上皮细胞系中α -防御素5的表达受microrna调控

D. Miles, Jun Shen, A. Chuang, Fenshi Dong, Feng Wu, J. Kwon
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引用次数: 13

摘要

在炎症性肠病(IBD)中,不适当的免疫反应导致慢性粘膜炎症。这种反应可能部分是由于防御素的失调,这是内源性产生的抗菌肽。本研究确定了microRNAs (miRNAs)是否调控α-防御素5 (DEFA5),这可能进一步影响两者在IBD发病机制中的作用。方法在Caco-2细胞中检测DEFA5 mRNA和蛋白的诱导作用。计算机分析确定了DEFA5的推测miRNA结合位点。在Caco-2细胞中评估这些mirna的表达。通过荧光素酶测定这些mirna对DEFA5表达的调节。用miR-124和miR-924模拟物转染Caco-2细胞,检测DEFA5 mRNA和蛋白的表达。结果Caco-2细胞可诱导DEFA5 mRNA及蛋白表达。在DEFA5中发现了15个推测的miRNA结合位点。诱导后miR-124和miR-924的表达降低。与转染空载体相比,转染含有DEFA5 miRNA结合位点的荧光素酶构建体导致荧光素酶活性降低。转染含有不匹配的miRNA结合位点的报告构建体导致荧光素酶活性的恢复。转染miRNA可模拟DEFA5 mRNA表达和蛋白表达的降低。结论miR-124和miR-924负向调控DEFA5 mRNA和蛋白的表达。这些数据暗示了mirna在肠道先天免疫调节和IBD发病机制中的作用。
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Alpha-Defensin 5 Expression is Regulated by microRNAs in the Caco-2 Intestinal Epithelial Cell Line
Background In inflammatory bowel disease (IBD), an inappropriate immune response leads to chronic mucosal inflammation. This response may be partly due to dysregulation of defensins, which are endogenously produced antimicrobial peptides. This study determined whether microRNAs (miRNAs) regulate α-defensin 5 (DEFA5), which could further implicate both in IBD pathogenesis. Methods Induction of DEFA5 mRNA and protein expression was determined in Caco-2 cells. An in silico analysis identified putative miRNA binding sites of DEFA5. Expression of these miRNAs was assessed in Caco-2 cells. Regulation of DEFA5 expression by these miRNAs was measured by luciferase assays. Caco-2 cells were transfected with miR-124 and miR-924 mimics, and DEFA5 mRNA and protein expression was measured. Results DEFA5 mRNA and protein expression was inducible in Caco-2 cells. Fifteen putative miRNA binding sites were found in DEFA5. The expression of miR-124 and miR-924 decreased following induction. Transfection of a luciferase construct containing the DEFA5 miRNA binding sites resulted in a decrease in luciferase activity compared to transfection of the empty vector. Transfection of a reporter construct containing mismatched miRNA binding sites resulted in restoration of luciferase activities. Transfection of miRNA mimics decreased DEFA5 mRNA expression and protein expression. Conclusions miR-124 and miR-924 negatively regulate DEFA5 mRNA and protein expression. These data implicate miRNAs in intestinal innate immune regulation and IBD pathogenesis.
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