Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic cell intermediates and pulmonary fibrosis [eCLIP-seq]

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation to safeguard the lung from spontaneous alveolar destruction and fibrosis. Using mice and organoid models with genetic ablation of let-7a1/let-7f1/let-7d cluster (let-7afd) in AT2 cells, we demonstrate prevents AT1 differentiation and results in aberrant accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP) with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2/MYC which drives an aberrant fibrotic cascade. Additional analyses using CUT&RUN-sequencing revealed an important epigenetic role of let-7 in promotion of histone acetylation and methylation, thereby reprogramming AT2 cells into fibrogenic ADIs. Enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP+let7) of chimeric miRNA-mRNA complexes from lungs of wild-type mice following 6-days of bleomycin treatment.

对肺2型肺泡（AT2）祖细胞的研究已明确，其在肺修复与疾病进程中可分化为1型肺泡细胞（AT1细胞），相关核心机制已得到揭示。然而，调控这一分化过程的微小RNA（miRNA）介导的转录后调控机制，目前仍未得到充分探究。本研究证实，let-7微小RNA家族在维持AT2细胞静息状态中发挥稳态调控作用：一方面可抑制AT2过渡细胞的异常蓄积，另一方面能促进AT1细胞分化，从而保护肺部免受自发性肺泡破坏与纤维化损伤。我们通过构建AT2细胞中敲除let-7a1/let-7f1/let-7d基因簇（let-7afd）的小鼠及类器官模型，证实该基因簇缺失会阻断AT1细胞分化，并导致AT2过渡细胞异常蓄积，进而引发进行性肺纤维化。将增强型AGO2紫外交联免疫沉淀测序（AGO2-eCLIP）与AT2细胞的RNA测序数据整合分析后，我们发现let-7的直接靶基因参与了以BACH1/EZH2/MYC为核心的癌基因前馈调控网络，该网络可驱动异常纤维化级联反应。额外的CUT&RUN测序分析显示，let-7在调控组蛋白乙酰化与甲基化过程中发挥重要表观遗传作用，由此将AT2细胞重编程为致纤维化疾病相关肺泡细胞（ADIs）。本研究还对博莱霉素处理6天后的野生型小鼠肺部的嵌合miRNA-mRNA复合物，开展了增强型AGO2紫外交联免疫沉淀测序（AGO2-eCLIP+let7）。