(433) Differential small RNA expression analysis reveals TGF-β role in SARS-CoV-2 infection of the CF airway

Background: SARS-CoV-2 induces humoral immune response controlled by cytokines, including TGF-β and regulates TGF-β-mediated gene expression. TGF-β1 is secreted by T regulatory cells (Tregs), dendritic cells, and airway epithelial cells during SARS-CoV-2 lung disease and modulation of TGF-β signaling by viral infections can lead to pulmonary fibrosis. Two proteins found to play a role in both CF and COVID-19 are angiotensin-converting enzyme 2 (ACE2) and serine protease transmembrane serine protease 2 (TMPRSS2). The entry of the SARS-COV-2 into the cell is dependent upon binding to ACE2 and cleavage by TMPRSS2. The regulation of TMPRSS2 and ACE2 can be mediated by micro(mi)RNAs, as effectors of TGF-β. We aimed to identify miRNAs and the target genes associated with COVID-19 pathogenesis in cellular model of CF lung disease in response to TGF-β1.

Methods: Parental CFBE41o- cells were used and results were validated in primary human bronchial epithelial (HBE) cells. For Small RNAseq, total RNA was used. Cluster generation and 75 bp single-read, single-indexed sequencing was performed on Illumina NextSeq 500’s. miRNA Differential Analysis was done by the edgeR package. Pathway analysis was done by Ingenuity Pathway Analysis software (QIAGEN Bioinformatics). The significantly upregulated and downregulated miRNA from differential analysis were used as query list for miRNA network analysis. The Ingenuity Pathway Analysis (IPA) from Qiagen for analyzing Covid-19 associated pathways affected by differentially regulated miRNAs. miRNAs predicted to target the Covid-19 associated genes were collected from TargetScan Human release 7.2, miRmap, Diana-TarBase v.8, and miRBase bioinformatics tools. Anti-miRNA oligonucleotide miRCURY LNA™ Power Inhibitors or control (Exiqon) were used.

Results: Total 48 miRNA were found dysregulated in vehicle vs. TGF-β1 treated CFBE41o- cells. We found 38 and 19 pathways uniquely affected by the upregulated and downregulated miRNAs, respectively. We found 43 miRNAs targeting 119 different mRNA of the proteins associated with coronavirus pathogenesis pathway. Next, to examine how these miRNAs are affecting the replication of coronavirus, we specifically selected coronavirus replication pathway and use the same strategy to build a network in IPA. We used path designer feature and found 21 miRNAs targeting 21 different mRNA of the proteins associated with coronavirus replication pathway. Next, we focused on miRNAs upregulated by TGF-β1, miR-136-3p and miR-369-5p targeting ACE2. We confirmed the small RNAseq results by qRT-PCR that TGF-β1 increased the miRNAs expression in CFBE41o- cells. Upregulation of the miRNAs was followed by inhibition of ACE2 mRNA and proteins levels and the effect was prevented by specific anti-miRNA oligonucleotides. Validation studies were performed in F508del-HBE cells in the presence of elexacaftor/tezacaftor/ivacaftor (Trikafta) or vehicle control. Confocal microscopy studies demonstrated specific pattern of subcellular localization of ACE2 enhanced by Trikafta in F508de-HNE cells. Studies outlined above were also performed to examine the role of miRNA-561-5p, downregulated by TGF-β1 on its target gene TMPRSS2.

Conclusions: miRNAs may be important effectors of TGF-β signaling that modulates SARS-CoV-2 invasion of the CF airway. Trikafta may have also modulate localization of the proteins critical for SARS-CoV-2 invasion.

Acknowledgements: This work was supported by NIH R01 HL144539 & CFF SWIATE18G0 (to A.S-U.); CFF RDP R883-15R0 (to C.B.)

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