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


METHODS
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 SiRNA iii 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.
Yan
Yanguo
Y
Department of Gastrointestinal and Pancreatic Surgery, The First Affiliated Hospital Research Center for Gastric Cancer, Sun Yat-sen University, Guangzhou 510080, China.
Zhan
Wenhua
W
Zhao
Gang
G
Ma
Jinping
J
Cai
Shirong
S
D000942:Antigens, Bacterial / Q000378:metabolism*; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018274:Electroporation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000382:microbiology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016209:Interleukin-8; D034741:RNA, Small Interfering; C080866:cagA protein, Helicobacter pylori