Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity

Abstract

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing “protective” drugs. This approach could also prove useful to other respiratory pathologies.

TNFRSF1B Signaling Blockade Protects Airway Epithelial Cells from Oxidative Stress

“…A high-throughput RNAi screening for oxidative stress susceptibility genes acting in CF airway epithelial cells yielded 167 siRNA sequences able to induce H 2 O 2 -mediated oxidative stress resistance in 6CFSMEo cells and 4452 putative targets with unique Entrez identifiers ( Table S1 , see “Material and Methods” for details) [ 8 ]. Thus, we focused on a particular siRNA sequence targeting the TNFRSF1B (TNFR2) transcript ( Figure 1 A) according to the following evidence: (i) this siRNA presented a high percentage of continuous matching homology with the TNFRSF1B gene (73%, according to Basic Local Alignment Search Tool (BLAST) analysis) ( Figure 1 B); (ii) its function as a putative death receptor [ 10 , 20 , 21 ] and the implication of the TNF-TNFR pathway as an important hub regulated by ROS in inflammation [ 22 ].…”

“…The siRNA sequence against TNFRSF1B (clone 178; Figure 1 A) was previously obtained through a high-throughput RNAi screening using a randomized siRNA library aimed to identify oxidative stress susceptibility genes [ 8 ]. The referred sequence introduced within the human U6 and H1 RNA polymerase III promoters arranged in opposite orientations [ 13 ] was amplified by proofreading the polymerase chain reaction (PCR) through “ClaI/NotI F” and “EcoRI-H1 R” primers ( Table 1 ), cloned into the pJET1.2 vector (Thermo Fisher Scientific) obtaining pJET1.2-siRNA ( Figure 1 ), and sequence verified (STAB VIDA, Caparica, Portugal) using “pJET 1.2 F” and “pJET 1.2 R” primers ( Table 1 ).…”

“…The corresponding transfection complexes formed after 45 min met with 2 × 10 5 cells per well added dropwise, in a final volume of 220 μL. This mixture was removed after 48 h, and the wells were refilled with an equal volume of 0.3 mM H 2 O 2 [ 8 ]. After 6 h of cell exposure to H 2 O 2 , the oxidant was removed, 100 μL/well of Alamar Blue (AB) reagent (Bio-Rad, Hercules, CA, USA) was included, and three spectrophotometer readings were performed at 19 h, 24 h, and 48 h to monitor cell viability (PowerWave XS, BioTek, Winooski, VT, USA).…”

“…The capacity to confer resistance to oxidative stress was assessed for the siRNA sequence targeting the TNFRSF1B transcript (siRNA 178) obtained in the initial screen through descriptive statistics and using a mixed-effects model, as previously described [ 8 ].…”

“…In a previous study, we performed a genome-wide RNA interference (RNAi) screening using a randomized small interfering RNA (siRNA) library to identify oxidative stress susceptibility genes/pathways in CF submucosal gland epithelial cells [ 8 ]. We unveiled the involvement of the tumor necrosis factor (TNF) pathway in oxidative stress and turned to a particular screening hit, the tumor necrosis factor receptor superfamily member 1B ( TNFRSF1B ; TNFR2 ) gene (Gene ID: 7133) [ 9 ].…”