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Knockdown of interleukin-8 in airway epithelial cells.pdf (618.18 kB)

Knockdown of interleukin-8 in airway epithelial cells

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journal contribution
posted on 2023-07-31, 14:28 authored by Fatema Mewa, Catherine GreeneCatherine Greene

Introduction: Cystic fibrosis (CF) is a neutrophil-dominated lung disease. The neutrophil chemokine interleukin-8 (IL-8) is present at higher than normal levels in the lungs of individuals with CF and is a key factor responsible for neutrophil infiltration into the lungs. Lipopolysaccharide (LPS) expressed by Pseudomonas aeruginosa in the CF lung can stimulate IL-8 expression by airway epithelial cells. Inhibiting the expression of IL-8 using small-interfering RNA (siRNA) represents a potential gene therapy approach for CF. Here, the efficacy of an IL-8-directed siRNA at inhibiting IL-8 gene and protein expression is tested in basal and LPS-stimulated human bronchial epithelial cells.

Methods: 16HBE14O- cells were transfected with either scrambled, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or IL-8-specific siRNAs. RNA was then isolated and used in quantitative real-time PCR to measure GAPDH, IL-8 or beta-actin (housekeeping) gene expression. Lastly, IL-8 protein production was quantified in cell supernatants by IL-8 ELISA.

Results: Transfection of 16HBE14O- cells with 30nM IL-8 siRNA for 48 hours led to an 84% knockdown in IL-8 mRNA (p<0.05). Treatment with LPS (10μg/ml, 6h) increased IL-8 gene expression 1.5-fold (p<0.05) and IL-8 siRNA caused a significant knockdown of IL-8 protein in LPS-treated cells (p<0.05). Furthermore, basal IL-8 protein production was decreased by IL-8 siRNA. LPS treatment increased IL-8 protein expression in 16HBE14O- cells. IL-8 siRNA failed to decrease this effect.

Conclusions: The data show that IL-8 siRNA can inhibit basal and LPS-induced IL-8 gene expression in 16HBE14O- cells. Further investigation is required to optimise the conditions for inhibition of IL-8 protein production, and to study its potential role in CF therapy.

Funding

Health Research Board

History

Comments

The original article is available at http://www.rcsismj.com/ Part of the RCSsmj collection: https://doi.org/10.25419/rcsi.c.6767511.v1

Published Citation

Mewa F, Greene C. Knockdown of interleukin-8 in airway epithelial cells. RCSIsmj. 2012;5(1):18-23

Publication Date

2012

Department/Unit

  • Beaumont Hospital
  • Medicine
  • Undergraduate Research

Publisher

RCSI University of Medicine and Health Sciences

Version

  • Published Version (Version of Record)