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Abstract: FR-PO019

Transforming Growth Factor-β Regulates Angiotensin Converting Enzyme 2 by MicroRNA Mediated Mechanism

Session Information

Category: Coronavirus (COVID-19)

  • 000 Coronavirus (COVID-19)

Authors

  • Hejenkowska, Ewelina, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Mitash, Nilay, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Donovan, Joshua, University of Virginia School of Medicine, Charlottesville, Virginia, United States
  • Bertrand, Carol A., University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Swiatecka-Urban, Agnieszka, University of Virginia School of Medicine, Charlottesville, Virginia, United States
Background

TGF-β signaling plays an essential role in tissue fibrosis and mediates profibrotic programs after SARS-CoV-2 infection in the kidney and lung. SARS-CoV-2 also induces humoral immune responses controlled by cytokines, including TGF-β. Studies have found that the incidence of SARS-CoV-2 infection and the severity of Covid-19 in cystic fibrosis (CF) patients is lower than the general population. We studied how SARS-CoV-2 regulates TGF-β-mediated gene expression in the CF airway.

Methods

Small RNAseq was performed in human bronchial epithelial cells CFBE41o- from a patient homozygous for the F508del mutation in the CFTR gene on Illumina NextSeq 500’s. Pathway analysis was done by Ingenuity Pathway Analysis (IPA) software (QIAGEN) and miRNet browser. IPA was used for analyzing coronavirus associated pathways affected by differentially regulated miRNAs. miRNAs predicted to target the coronavirus 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. Cells expressing F508del or wild type CFTR were used to compare the results in CF and non-CF models. Findings were validated in primary human bronchial epithelial (HBE) cells.

Results

Compared to vehicle control, TGF-β1 dysregulated 48 miRNAs; 38 and 19 pathways were 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 and 21 miRNAs targeting 21 different mRNA of the proteins associated with coronavirus replication pathway. Two miRNAs upregulated by TGF-β1 target the host receptor for SARS-CoV-2 invasion, angiotensin converting enzyme 2 (ACE2). We confirmed the results by qRT-PCR that TGF-β1 increased expression of specific miRNAs targeting ACE2 mRNA. Upregulation of the miRNAs was followed by inhibition of ACE2 mRNA and protein levels and the effect was blocked by specific anti-miRNA oligonucleotides. The above results differed between the CF and non-CF cells.

Conclusion

miRNAs may be important effectors of TGF-β modulating SARS-CoV-2 pathogenicity and replication in the CF airway. Ongoing studies focus on elucidating the mechanisms of SARS-CoV-2 invasion of kidney cells.

Funding

  • Other NIH Support