TGF-ß1 alter the SARS-CoV2 pathogenesis and modulates ACE2 expression by miRNA dependent mechanism

We studied miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in CF airway epithelial cell models in response to TGF-ß1. Small RNAseq in CF human bronchial epithelial cell line treated with TGF-ß1 and miRNA profiling characterized TGF-ß1 effects on the SARS-CoV-2 pathogenesis pathways. Among the effectors, we identified and validated two miRNAs targeting ACE2 mRNA using different CF and non-CF human bronchial epithelial cell models. We have shown that TGF-ß1 inhibits ACE2 expression by miR-136-3p and miR-369-5p. ACE2 levels were higher in cells expressing F508del-CFTR, compared to wild-type(WT)-CFTR and TGF-ß1 inhibited ACE2 in both cell types. The ACE2 protein levels were still higher in CF, compared to non-CF cells after TGF-ß1 treatment. TGF-ß1 prevented the functional rescue of F508del-CFTR by ETI in primary human bronchial epithelial cells while ETI did not prevent the TGF-ß1 inhibition of ACE2 protein. Finally, TGF-ß1 reduced binding of ACE2 to the recombinant monomeric spike RBD. Our results may help to explain, at least in part, the role of TGF-ß1 on the SARS-CoV-2 entry via ACE2 in the CF and non-CF airway. Overall design: Parental human bronchial epithelial CFBE41o- cells, were maintained in Minimum Eagle's Medium (MEM; Thermo Fisher Scientific, Walthman, MA, USA) supplemented with 50 units/ml penicillin, 50 µg/ml streptomycin and 2mM L-glutamine (Thermo Fisher Scientific), and 10% fetal bovine serum (FBS; Corning, NY) in a 5% CO2, 95% air incubator at 37°C. To establish polarized monolayers, cells were seeded on 24 mm diameter Transwell filters (Corning, New York, NY) at 2×106 and grown in air-liquid interface (ALI) culture at 37 °C for 6-9 days. Human TGF-ß1 (Sigma-Aldrich, St. Louis, MO, USA)(n=3), or vehicle control (4 mM HCl + 1 mg/mL Bovine Serum Albumin; BSA, R&D Systems)(n=3) was used at concentration 15 ng/mL