Use of SiRNA to investigate the role of CagA on H. pylori induced IL-8 production from gastric epithelial cells.

摘要

BACKGROUND/AIMS: H. pylori infection can stimulate gastric epithelial cells to release IL-8. We studied the effect of small interference RNA (SiRNA) on CagA expression and the H. pylori-induced release of IL-8 by gastric epithelial cells. METHODOLOGY: SiRNA's were transferred into CagA positive strain H. pylori NCTC 11637 using electroporation. The CagA positive strain NCTC 11637 and the CagA negative strain NCTC 11639 were co-cultured with gastric epithelial cells and the level of IL-8 in the supernatant was measure by ELISA. RT-PCRs and Western blot were performed to detect CagA expression. RESULTS: The level of IL-8 induced by NCTC 11637 was higher than that produced by infection with NCTC 11639. Treatment with SiRNA iii or v resulted in a decrease in IL-8 production of more than 50% and a corresponding decrease in CagA mRNA. The levels of CagA mRNA at 6, 12, and 24 hours after electroporation were 31.3%, 43.5%, and 76.8% of the control levels, respectively (P = 0.0025). Western blot following SiRNAiii showed the level of CagA protein degraded to 30% of control by x hours. CONCLUSIONS: CagA is involved in H. pylori induced IL-8 release from gastric epithelial cells.