Elevated <i>Mirc1/Mir17-92</i> cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the <i>CF transmembrane conductance regulator (CFTR)</i> gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (<i>Mirs</i>), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of <i>Mirs</i> in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the <i>Mirc1/Mir17-92</i> cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between <i>Mirc1/Mir17-92</i> and autophagy gene expression. An <i>in silico</i> study for targets of <i>Mirs</i> that comprise the cluster suggested that the majority of the <i>Mirs</i> target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated <i>Mirs</i>, via restoration of autophagy. In vivo, downregulation of <i>Mir17</i> and <i>Mir20a</i> partially restored autophagy expression and hence improved the clearance of <i>Burkholderia cenocepacia</i>. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

囊性纤维化（Cystic fibrosis, CF）是一种致命的遗传性疾病，会严重累及肺部，其直接致病原因为囊性纤维化跨膜传导调节因子（CF transmembrane conductance regulator, CFTR）基因发生突变，导致CFTR蛋白功能缺陷。巨自噬/自噬（Macroautophagy/autophagy）是一种高度调控的生物学过程，可在应激与饥饿状态下为细胞提供能量，并能清除巨噬细胞内的病原体与功能异常的蛋白质聚集物。然而，CF患者与CF模型小鼠体内的该过程存在缺陷，其巨噬细胞的自噬活性受到抑制。对微小RNA（microRNAs, Mirs）及其他非编码RNA的研究持续为治疗靶点开发提供新方向。本研究旨在阐明微小RNA在CF巨噬细胞自噬相关基因失调中的作用，并通过靶向这些微小RNA以恢复宿主防御功能、改善CFTR通道功能。我们发现，与野生型（wild-type, WT）巨噬细胞相比，CF巨噬细胞的自噬相关蛋白表达水平降低，而Mirc1/Mir17-92基因簇的表达水平升高，提示该基因簇可能作为自噬的负调控因子。该基因簇的缺失或表达下调可升高自噬相关蛋白的表达水平，表明Mirc1/Mir17-92与自噬基因的表达呈典型的负相关关系。针对该基因簇所含微小RNA的计算机模拟分析（in silico）靶标预测显示，绝大多数此类微小RNA均可靶向结合自噬相关mRNA，该结果通过荧光素酶报告基因实验得到了验证。值得注意的是，携带突变型F508del CFTR的巨噬细胞，其细胞膜的卤化物转运能力受损，而通过下调这些本就高表达的微小RNA、恢复自噬功能，可修复该转运缺陷。在体内实验中，下调Mir17与Mir20a的表达可部分恢复自噬相关蛋白的表达，进而改善了机体对洋葱伯克霍尔德菌（Burkholderia cenocepacia）的清除能力。综上，本研究加深了我们对CF发病机制的理解，并为CF患者恢复CFTR功能与自噬活性提供了全新的治疗策略。

搜集汇总

背景与挑战

背景概述

该数据集聚焦于囊性纤维化（CF）疾病中巨噬细胞的自噬调控机制，研究发现Mirc1/Mir17-92集群表达升高会负调控自噬和CFTR功能，通过下调这些microRNAs可恢复自噬并改善细菌清除能力。数据集提供了相关实验数据和补充材料，支持CF病理生物学机制的理解，并为治疗提供了新靶点。