Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Abstract

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr -/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells: potential roles in airway inflammation

“…After incubation for 4 h, cells were transduced with a viral vector at 2 500 particles/cell (or 50 moi) in culture media without serum. We usually achieved nearly 100% transduction at this vector concentration [57,59]. After 2 h, fetal bovine serum was added to a final concentration of 10%.…”

“…EMSAs were performed using double-stranded oligonucleotide probes end-labeled with [g-32 P]-ATP by T4 polynucleotide kinase or [α-32 P]-dCTP by Klenow for DNA fragments. For NF-κB EMSAs [57], a labeled probe (20 000 cpm/reaction) was incubated with 5 µg of nuclear extracts from BEAS-2B cells in 20 µl solution consisting of 10 mM Tris (pH 8.0), 15 mM KCl, 1 mM EDTA, 5% glycerol, 1 mM DTT, 1 mg BSA, 1 µg poly(dI-dC) at room temperature for 20 min. For Ets EMSAs, the labeled probes were incubated each with 2.5 µg of purified Ese-1 recombinant protein.…”

“…The design of the mouse U6-promoter-driven shRNA expression cassettes and viral vector was described by Gonzales-Santos et al [59]. The viral vector was produced and purified as described [57].…”

Inhibition of BCNU-Resistance in Glioma Cell Lines by Small Interfering Rna Targeting to Interleukin-8 Gene

“…It has been demonstrated that RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells [22,23]. Besides, it was demonstrated that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells [24]. In this research, to explore the effect of IL-8 on BCNU resistance, siRNAs targeting IL-8 was used for inhibiting the IL-8 expression in BCNU resistant cell lines SWOZ1 and SWOZ2-BCNU.…”

From sequence to function: using RNAi to elucidate mechanisms of human disease

“…Retroviral-, 48,49,51,52 adenoviral-, 53,54 and lentiviral-based 50,55 systems have been utilized by numerous groups with a high success rate. As lentiand adenovirus are capable of infecting both dividing and nondividing cells, transduction of shRNA via these viruses may be the only feasible way to achieve knockdown in extremely slow-growing and nondividing cell types.…”