Abstract Introduction CFTR expression varies among epithelial cell types: highest in ionocytes, modest in basal and secretory cells and lowest in ciliated cells. Regulatory mechanisms underlying differential CFTR expression across these epithelial subpopulations remain unknown. Non-coding microRNAs modulate gene expression, with TGF-β dependent miR-145 particularly relevant to CFTR. In the present work, we used VISION (v3.0.0) package in R to understand the TGF-β signaling pathway in CF at a single cell level and compared with CFTR expression in epithelial subtypes. Hypothesis The TGF-β/miR-145 axis regulates CFTR expression in specific epithelial cell subtypes. Methods To understand TGF-β signaling across epithelial cells, we analyzed scRNA-Seq data (GSE150674) for TGF-β relevant genes using VISION. Vision runs on single cell manifold from scRNA-Seq data and uses an autocorrelation statistic to calculate pathway signature score for individual cells. To analyze miR-145 expressions in epithelial subtypes, we employed two orthogonal approaches: a) PrimeFlowTM to detect in-situ microRNA levels and b) flow-sort epithelial cell types and analyzed miR-145 using qPCR. Epithelial subtypes were analyzed using flow cytometry with antibodies specific to basal (CD172), secretory (CD66c), ionocytes (BSND) and ciliated cells (acetylated tubulin). We coupled miR-145 PrimeflowTM assay with imaging flow cytometry to detect its expression at single cell spatial resolution. Results TGF-β (signaling) pathway signature scores were higher in CF compared to non-CF (CO) cells (CF: 0.144±0.001 vs 0.14±0.0001 in CO, p-value 0). In epithelial subtypes, basal (0.17±0.001) and ionocytes (0.2±0.01) showed higher TGF-β scores than ciliated (0.11±0.001) and secretory cells (0.1±0.001). In relation to CFTR expression across epithelial cells, TGF-β scores were decreased inversely in cells with higher CFTR expression (High: 0.13±0.01, moderate: 0.2±0.007, low: 0.21±0.01, absent: 0.14±0.001). Moderate CFTR expressing cells in CF showed high TGF-β score compared to CO (0.2±0.007 vs 0.17±0.005 in CO, p-value 0). PrimeflowTM revealed a significant reduction in miR-145 mean-pixel intensity in ionocytes inversely related to CFTR expression (CFTR-high: 2275±267.5, CFTR-low: 918.8±130.6, p 0.03). In sorted epithelial cells, CFTR expression was increased in ionocytes ( 10000-fold) but decreased in secretory (0.6-fold) and ciliated cells (0.75-fold). Conversely, miR-145 expression was decreased in ionocytes (0.23-fold) but similar in both secretory (1.36-fold) and ciliated (1.08-fold) cells compared to basal cells. Conclusions TGF-β signaling is inversely associated with CFTR expression across epithelial cells, with similar associations in miR-145. Our findings suggest that TGF-β dependent miRNA may regulate CFTR expression in epithelial subpopulations and provide a cell-specific target to enhance CFTR function. This abstract is funded by: KPRI, Cystic Fibrosis Foundation
Kumar et al. (2026) studied this question.