Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic cell intermediates and pulmonary fibrosis [RNA-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. Overall design: Fresh flow sorted let-7afd knockout and control lung murine AT2 cells obtained from conditional AT2 knockout mice were examined by RNA-seq. The AT2 cells were collected from pools of three mice per group (n=3) following 6-days of tamoxifen administration. Whole lungs were examined by RNA-seq from conditional AT2 specific let-7afd knockout and control mice following 6-days of tamoxifen. The whole lungs were collected from three mice of each genotype. Whole lungs were examined by RNA-seq from conditional AT2 specific let-7afd knockout and control mice following 6-months of booster tamoxifen. The whole lungs were collected from four mice of each genotype. Let-7afd knockout and control lung murine AT2 cell 3D organoids cultures were grown in AMM media for 14-days and then examined by RNA-seq. The organoids were collected from two mice of each genotype following 6-days of tamoxifen.