Effect Of Helicobacter Pylori On Morphology Of Human Gastric Cancer SGC-7901 Cells

Since Warren and Marshall et al isolated Helicobacter pylori(H. pylori,Hp)in stomach sinus from gastritis and peptic ulcer patient for the first time in 1982,because H.pylori infection has been closely related to digestive tract disease,a lot of studies have been done on its pathogenic mechanism.Helicobacter pylori is a spiral,gram-negative and microaerophilic bacterium,is one of the major factors inducing pathological changes in human gastric mucosa,and about half of people in the world are infected with it.Cytotoxin-associated gene A(cagA)is the key virulence factor of Hp,other virulence factor include VacA,cagT, cagE etc.Pathological and clinical studies have convincingly proved the etiological role of Hp in the development of chronic gastritis,peptic ulcer and gastric adenocarcinoma,but the mechanism of how Hp infection leads to gastroduodenal diseases always remains unclear.Small G protein RhoA is one of the important members of Rho family and plays an important role in the cell signal pathway.It has participated in a series of biological process,including the changes of cell shape,movement,regulation of cytoskeleton,shrink of smooth muscle and progression of tumor.The abnormal expression of RhoA is strongly associated with the occurrence and development of tumor.SHP-2 is a Src homology-2(SH2)domain-containing phosphatase.Most previous research concentrated on its influence on the signal pathway such as Rho family,MAPK and Ras et al.Recent researches have found that SHP-2 is also associated with many tumors.The subject was designed to study the effect of Helicobacter pylori on expression of SHP-2 and morphology,motility and cytoskeleton of human gastric cancer SGC-7901 cells and to find whether there is relevance between Helicobacter pylori and gastric disease.In this study,H.pylori was isolated from human gastric mucosa and cultured on solid culture plate.We starved the human gastric cancer cell line SGC-7901 for 24 h with cultrue media DMEM without serum.After stimulating the SGC-7901 cells with the sonicating extract for 1 hour,we observed the level of SHP-2 with semi-quantitative PCR(RT-PCR).The PCR products were then cloned and sequenced.We found that the mRNA level of SHP-2 in the SGC-7901 cells had no change before and after H.pylori stimulating.The sequencing results showed that they were 376 bp in length,when compared with the sequences in GenBank,they showed 93%in homology.Then the SGC-7901 cells were treated with the sonicating extract to observe the morphological alteration,cell motility and the cytoskeleton.Cell motility was examined by Boyden chamber motility assay and scrape-motility assay.Cytoskeleton was observed by F-actin phalloidin fluorescent staining.The results showed that the sonicating extract could enhance SGC-7901 cell motility,induce the appearance of hummingbird-phenotype,and increase cytoskeleton. The cytoskeleton in the cells transfected with wild type pcDNA RhoA were increased compared to the control cells;while mutant pcDNA RhoA had no effect on the cytoskeleton.So,we indicate that H.pylori sonicating extract has no effect on the mRNA level of SHP-2 in SGC-7901 cells;but it can provoke the morphological changes of the cells,such as enhancing SGC-7901 cells motility,inducing the appearance of hummingbird-phenotype,and increasing the cytoskeleton. The active form of RhoA plays an important role during this process.