HIF1A-dependent induction of alveolar miR-147b dampens SARS-CoV-2-associated ARDS via direct targeting of viral RNA

Abstract

BACKGROUND: Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality during viral pneumonia, including SARS-CoV-2 infections. Nevertheless, most patients with SARS-CoV-2 infections recover seamlessly without developing ARDS, suggesting the existence of endogenous pathways to protect the lungs. Since microRNAs (miRNAs) can regulate endogenous molecular pathways involved in lung protection, we hypothesized that alveolar miRNAs could function to dampen SARS-CoV-2-associated lung injury. METHODS: Screening studies in human alveolar epithelial cells and SARS-CoV-2-infected mice were performed to identify miRNAs induced during infection. Candidate miRNAs were confirmed via RT-qPCR. The role of hypoxia-inducible factor 1A (HIF1A) in regulating miRNA expression was examined in molecular studies. Loss- and gain-of-function approaches in a murine SARS-CoV-2 ARDS model were used to assess the physiological relevance of miRNA. Viral sequence analyses and site-directed mutagenesis were used to determine direct miRNA - viral RNA interactions. RESULTS: Screening studies identified miR-147b (hsa-miR-147b-3p or mmu-miR-147-3p) as the leading candidate during infection of human alveolar epithelia or mice with SARS-CoV-2. Functional and molecular studies implicate HIF1A in miR-147b induction during alveolar injury or SARS-CoV-2 infection. Studies in mice with induced deletion of miR-147b in alveolar epithelia (miR147fl/fl SPC-CreER mice) or nano-particle-mediated miR-147b overexpression revealed a protective role of alveolar-expressed miR-147b during murine SARS-CoV-2-associated ARDS. Moreover, we identified the ORF8 region within the SARS-CoV-2 template strand as a direct target of miR-147b, with an ORF8 silent mutation of the SARS-CoV-2 miR-147b-binding site abolishing the observed protection in vitro and in vivo. CONCLUSIONS: Alveolar epithelial cell-derived miR-147b serves as an endogenous lung protective miRNA against SARS-CoV-2-associated ARDS by directly targeting virus-encoded RNA, revealing a previously unrecognized antiviral and lung-protective mechanism.