DataSheet_1_Extracellular Vesicle Associated miRNAs Regulate Signaling Pathways Involved in COVID-19 Pneumonia and the Progression to Severe Acute Respiratory Corona Virus-2 Syndrome.zip

BackgroundExtracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19.
MethodsWe prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS.
ResultsDGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted<0.001), among others, targeting interleukin-6 (IL6) (25.1, 15.2 - 88.2 pg/ml in COVID-19 pneumonia) and OR52N2, an olfactory smell receptor in the nasal epithelium. In contrast, miR-3168 was significantly downregulated in COVID-19 ARDS (log2FC=-2.13, padjusted=0.003) and targeted interleukin-8 (CXCL8) in a completely activated network. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p.
ConclusionEV-derived miRNAs might have important regulative functions in the pathophysiology of COVID-19: CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. These miRNAs may serve as biomarkers or as possible therapeutic targets.

背景：细胞外囊泡（Extracellular vesicles, EVs）是炎症性肺部疾病中细胞间通讯的介质，可作为微小RNA（miRNAs）的载体，这些miRNAs被受体细胞摄取后，可调控信使RNA（mRNA）转录本及信号通路。本研究旨在探讨循环EVs相关的miRNAs是否可调控新型冠状病毒肺炎（COVID-19）中的免疫过程。
方法：本研究前瞻性纳入20例有症状的COVID-19肺炎患者、20例重症COVID-19（严重急性呼吸综合征冠状病毒2综合征，急性呼吸窘迫综合征，ARDS）机械通气患者及20例健康对照者。采用沉淀法分离EVs，提取总RNA，通过小RNA测序（small RNA sequencing）进行表达谱分析，并通过差异基因表达分析（differential gene expression analysis, DGE）进行评估。对组间差异表达的miRNAs进行生物信息学分析，预测其mRNA靶转录本并构建信号网络，以此分别比较COVID-19肺炎与健康状态、COVID-19肺炎与重症COVID-19 ARDS之间的差异。
结果：与健康对照相比，COVID-19肺炎患者中共有43个差异表达显著的miRNAs（其中25个表达下调）；与COVID-19肺炎患者相比，重症COVID-19 ARDS患者中有20个差异表达miRNAs（其中15个表达下调）。对比COVID-19肺炎与健康对照的网络分析显示，miR-3168表达上调（log₂FC=2.28，校正后P值<0.001），其靶基因为白细胞介素6（interleukin-6, IL6，COVID-19肺炎患者中其水平为15.2~88.2 pg/ml，均值约25.1 pg/ml）及鼻上皮嗅觉受体OR52N2。与之相反，miR-3168在重症COVID-19 ARDS患者中表达显著下调（log₂FC=-2.13，校正后P值=0.003），且在完全激活的网络中靶向趋化因子配体8（CXCL8）。Toll样受体4（Toll-like receptor 4, TLR4）在COVID-19肺炎中被miR-146a-5p抑制，而在ARDS中被let-7e-5p上调。
结论：EV来源的miRNAs可能在COVID-19的病理生理过程中发挥重要调控功能：CXCL8可招募中性粒细胞进入肺部，造成上皮损伤；而与严重急性呼吸综合征冠状病毒2（severe acute respiratory corona virus-2, SARS-CoV-2）刺突蛋白紧密结合的激活型TLR4，可上调细胞表面血管紧张素转换酶2（ACE2）的表达，并破坏分泌肺表面活性物质的II型肺泡细胞，二者均可导致肺毛细血管渗漏及ARDS。这些miRNAs或可作为生物标志物或潜在治疗靶点。