1 Respiratory Research Division, Department of Medicine, and.
Am J Respir Crit Care Med. 2014 Feb 1;189(3):263-73. doi: 10.1164/rccm.201306-1151OC.
Retention of abnormal α1-antitrypsin (AAT) activates the unfolded protein response in AAT-deficient monocytes. The regulatory role of microRNAs (miRNAs) in unfolded protein responses and chronic obstructive pulmonary disease pathogenesis has not been investigated.
To investigate miRNA expression and function in MM and ZZ monocytes and identify miRNA(s) regulating the unfolded protein response.
Peripheral blood monocytes were isolated from asymptomatic and symptomatic MM and ZZ individuals for miRNA expression profiling and pyrosequencing analysis. miRNA/gene and protein expression was measured with quantitative polymerase chain reaction and Western blotting. Overexpression and inhibition studies were performed with pre-miR or anti-miR, respectively. Luciferase reporter genes were used to elucidate direct miRNA-target interactions. Inflammatory cytokines were detected using the Meso Scale Discovery Plex assays.
Forty-three miRNAs were differentially expressed, with miR-199a-5p most highly up-regulated in asymptomatic ZZ versus MM monocytes. miR-199a-2 promoter hypermethylation inhibits miR-199a-5p expression and was increased in symptomatic MM and ZZ monocytes compared with asymptomatic counterparts. GRP78, activating transcription factor 6, p50, and p65 were increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or up-regulation of these markers was observed after miRNA modulation. Direct miR-199a-5p targeting of activating transcription factor 6, p50, and p65 by miR-199a-5p was demonstrated using luciferase reporter systems. Overexpression of miR-199a-5p also decreased other arms of the UPR and expression of cytokines that are not putative targets.
miR-199a-5p is a key regulator of the unfolded protein response in AAT-deficient monocytes, and epigenetic silencing of its expression regulates this process in chronic obstructive pulmonary disease.
异常α1-抗胰蛋白酶（AAT）的保留会激活 AAT 缺乏的单核细胞中的未折叠蛋白反应。miRNAs（miRNA）在未折叠蛋白反应和慢性阻塞性肺疾病发病机制中的调节作用尚未得到研究。
研究 MM 和 ZZ 单核细胞中的 miRNA 表达和功能，并确定调节未折叠蛋白反应的 miRNA。
从无症状和有症状的 MM 和 ZZ 个体中分离外周血单核细胞，进行 miRNA 表达谱和焦磷酸测序分析。用定量聚合酶链反应和 Western 印迹法测量 miRNA/基因和蛋白质表达。分别用 pre-miR 或 anti-miR 进行过表达和抑制研究。使用荧光素酶报告基因阐明直接 miRNA-靶相互作用。使用 Meso Scale Discovery Plex 测定法检测炎症细胞因子。
有 43 个 miRNA 表达差异，无症状 ZZ 与 MM 单核细胞相比，miR-199a-5p 表达上调最明显。miR-199a-2 启动子超甲基化抑制 miR-199a-5p 表达，与无症状对照组相比，症状性 MM 和 ZZ 单核细胞中的 miR-199a-2 启动子超甲基化增加。与无症状 ZZ 单核细胞相比，症状性 ZZ 单核细胞中 GRP78、激活转录因子 6、p50 和 p65 增加。miRNA 调节后观察到这些标志物的相互下调或上调。荧光素酶报告系统证实，miR-199a-5p 直接靶向激活转录因子 6、p50 和 p65。miR-199a-5p 的过表达也降低了未折叠蛋白反应的其他分支和非假定靶标的细胞因子表达。
miR-199a-5p 是 AAT 缺乏的单核细胞中未折叠蛋白反应的关键调节因子，其表达的表观遗传沉默调节慢性阻塞性肺疾病中的这一过程。
