Cheng Zhe, Wang Xi, Dai Lingling, Jia Liuqun, Jing Xiaogang, Liu Ying, Wang Huan, Li Pengfei, An Lin, Liu Meng
Department of Respiratory and Critical Care Medicine, Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Oncotarget. 2017 Jul 1;8(40):67933-67941. doi: 10.18632/oncotarget.18913. eCollection 2017 Sep 15.
Injury to airway smooth muscle (ASM) cells hallmarks the pathological progression of asthma, a chronic inflammatory airway disease. MicroRNAs (miRNAs) play essential roles in the development of asthma as well as airway remodeling. Here we studied the involvement of miRNAs in the regulation of autophagic survival of ASM cells and airway disorder. We analyzed autophagy-associated factors LC3 and Beclin-1 by RT-qPCR and protein blotting in purified airway smooth muscle cells from ovalbumin (OVA)-induced asthmatic mice. The biological activity of miRNA targeting Beclin-1 was explored by bioinformatics method and confirmed in a dual luciferase reporter assay. Loss of function experiment was performed via transplantation of miRNA in OVA-induced asthmatic mice. We detected high autophagy levels in ASM cells, which appeared to result from augmentation of Beclin-1 protein, rather than Beclin-1 mRNA, suggesting presence of post-transcriptional control of Beclin-1. Next, miR-384 was figured out to be a Belcin-1-targeting miRNA, which significantly decreased after OVA treatment. Mechanistically, binding of miR-384 to 3'-UTR of Beclin-1 mRNA potently suppressed Beclin-1 protein translation in ASM cells, similar to previous finding in another cell type. In vivo, transplantation of miR-384 significantly attenuated Belcin-1 protein levels in ASM cells, resulting in reduced autophagy of ASM cells and attenuation of asthmatic features by OVA. Together, these data suggest that re-expression of miR-384 may reduce augmentation of Beclin-1-dependent autophagy of ASM cells, as a novel promising treatment for asthma.
气道平滑肌（ASM）细胞损伤是哮喘（一种慢性炎症性气道疾病）病理进展的标志。微小RNA（miRNA）在哮喘的发展以及气道重塑中发挥着重要作用。在此，我们研究了miRNA在ASM细胞自噬存活调节和气道紊乱中的作用。我们通过RT-qPCR和蛋白质印迹法分析了来自卵清蛋白（OVA）诱导的哮喘小鼠的纯化气道平滑肌细胞中自噬相关因子LC3和Beclin-1。通过生物信息学方法探索了靶向Beclin-1的miRNA的生物学活性，并在双荧光素酶报告基因检测中得到证实。通过在OVA诱导的哮喘小鼠中移植miRNA进行功能丧失实验。我们检测到ASM细胞中的自噬水平较高，这似乎是由于Beclin-1蛋白增加而非Beclin-1 mRNA增加所致，提示存在Beclin-1的转录后调控。接下来，发现miR-384是一种靶向Beclin-1的miRNA，在OVA处理后显著降低。机制上，miR-384与Beclin-1 mRNA的3'-UTR结合可有效抑制ASM细胞中Beclin-1蛋白的翻译，这与先前在另一种细胞类型中的发现相似。在体内，miR-384的移植显著降低了ASM细胞中Beclin-1蛋白水平，导致ASM细胞自噬减少以及OVA诱导的哮喘特征减轻。总之，这些数据表明，miR-384的重新表达可能减少ASM细胞中Beclin-1依赖性自噬的增加，这是一种有前景的哮喘新治疗方法。
