TGF-β1 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-β1. Small RNAseq in CF human bronchial epithelial cell line treated with TGF-β1 and miRNA profiling characterized TGF-β1 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-β1 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-β1 inhibited ACE2 in both cell types. The ACE2 protein levels were still higher in CF, compared to non-CF cells after TGF-β1 treatment. TGF-β1 prevented the functional rescue of F508del-CFTR by ETI in primary human bronchial epithelial cells while ETI did not prevent the TGF-β1 inhibition of ACE2 protein. Finally, TGF-β1 reduced binding of ACE2 to the recombinant monomeric spike RBD. Our results may help to explain, at least in part, the role of TGF-β1 on the SARS-CoV-2 entry via ACE2 in the CF and non-CF airway. 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-β1 (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

本研究针对转化生长因子-β1（TGF-β1）刺激下的囊性纤维化（cystic fibrosis, CF）气道上皮细胞模型中，影响严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）致病机制的微小RNA（miRNAs）及其基因靶点展开探究。通过对经TGF-β1处理的囊性纤维化人支气管上皮细胞系开展小RNA测序（small RNAseq）与miRNA表达谱分析，明确了TGF-β1对SARS-CoV-2致病通路的调控作用。在诸多效应分子中，我们借助多种囊性纤维化与非囊性纤维化人支气管上皮细胞模型，筛选并验证了两种靶向血管紧张素转换酶2（ACE2）信使RNA（mRNA）的miRNAs。研究证实，TGF-β1可通过miR-136-3p与miR-369-5p抑制ACE2的表达。相较于表达野生型（WT）-CFTR的细胞，表达F508del-CFTR的细胞内ACE2水平更高，且TGF-β1可在两种细胞系中均抑制ACE2的表达。经TGF-β1处理后，囊性纤维化细胞内的ACE2蛋白水平仍高于非囊性纤维化细胞。TGF-β1可抑制囊性纤维化跨膜传导调节因子（CFTR）矫正剂ETI对原代人支气管上皮细胞中F508del-CFTR的功能挽救作用，而ETI无法阻断TGF-β1对ACE2蛋白的抑制效果。此外，TGF-β1会降低ACE2与重组单体刺突蛋白受体结合域（receptor binding domain, RBD）的结合能力。本研究结果可在一定程度上解释TGF-β1通过调控ACE2通路，影响囊性纤维化与非囊性纤维化气道中SARS-CoV-2入侵的作用机制。亲本人支气管上皮CFBE41o-细胞培养于含50 U/ml青霉素、50 μg/ml链霉素、2 mM L-谷氨酰胺（均购自赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市）以及10%胎牛血清（fetal bovine serum, FBS，购自康宁公司，美国纽约州）的最低必需培养基（Minimum Eagle's Medium, MEM，Thermo Fisher Scientific，美国马萨诸塞州沃尔瑟姆市）中，于37℃、5% CO₂、95%空气的培养箱内培养。为建立极化单层细胞模型，将2×10⁶个细胞接种于直径24 mm的Transwell聚碳酸酯膜培养板（Corning，美国纽约州）中，于气液界面（air-liquid interface, ALI）培养体系中37℃培养6-9天。实验所用重组人TGF-β1（购自西格玛奥德里奇，美国密苏里州圣路易斯市，n=3）浓度为15 ng/ml，对照组为含4 mM HCl与1 mg/mL牛血清白蛋白（bovine serum albumin, BSA，购自R&D Systems）的溶剂对照（n=3）。