Identification of oxidative stress susceptibility-related genes in cystic fibrosis airway epithelial cells through functional genomics using a randomized siRNA library

Abstract

INTRODUCTION: Pulmonary manifestations are the main cause of morbidity and mortality in more than 90 % of cystic fibrosis (CF) patients who exceed the neonatal period. The chronic immune-inflammatory process, exacerbated by recurrent bacterial infections that induce an increase in oxidative stress, is a key component of the progressive destruction that occurs in their respiratory tract. Thus, the increase in reactive oxygen species due to the persistent activation of neutrophils, together with reduced anti-oxidant protection are the main contributors to oxidative stress and poor control of the immune-inflammatory response in CF patients.

OBJECTIVE: The main aim of this study is the identification and characterization of new genes involved in oxidative stress in CF human respiratory epithelial cells (oxidative stress- related CF modifying genes) using a randomized siRNA library.

MATERIAL AND METHODS: A high-throughput screening was performed using a randomized siRNA library introduced into the 6CFSMEo- CF bronchial epithelial cell line. Cells were then submitted to oxidative stress (H2O2). The genes whose silencing (inhibition of expression) induced resistance to H2O2-mediated apoptosis were identified.

RESULTS: After three successive screening rounds using a siRNA convergent expression module, 181 unique siRNAs sequences conferring oxidative stress resistance were obtained. From these, 167 were confirmed using a high-throughput reverse transfection technique and cell viability assay. By launching the 167 statistically significant oxidative stress-resistant siRNA sequences unveiled in the genetic screening against the Genebank database and contrasting them using the BLAST tool, we obtained a set of 444 siRNA target genes. The high-throughput silencing approach was validated confirming the relevance of two selected genes: TNFRSF1B and KCNQ1OT1. Data mining in public drug databases using the siRNA target genes obtained in the RNAi screen and its first interactors identified novel repurposable drugs that were able to induce resistance to oxidative stress in CF airway epithelial cells.

CONCLUSIONS: The introduction of a randomized library of siRNA in pulmonary epithelial cells and the subsequent large-scale screening of clones resistant to a lethal concentration of H2O2 allows the identification of new genes related to oxidative stress susceptibility in these cells through bioinformatic analysis. These analyses have indicated that the target genes are involved in pathways related to neuron term, RNA-splicing, oxidative stress and inflammation, cell cycle and metabolism, among others. Such functional genomics methodology, in addition to increasing the knowledge of the pathophysiology of CF respiratory epithelial cells, can contribute to the development of new therapeutic targets and both repurposable drug- and siRNA-based treatment strategies that would directly benefit CF patients.