| Background:Gastric cancer is one of the most common malignant tumors.Invasion and metastasis are important factors affecting the prognosis of gastric cancer.Helicobacter pylori infection is not only an important risk factor for gastric carcinogenesis,but it also is an important risk for the invasion and metastasis of gastric cancer.Recent studies indicate that actin cytoskeleton rearrangement is an important sign for tumor cell invasion and migration of tumor cells.H.pylori infection is known to induce the rearrangement of actin cytoskeleton in gastric epithelium cells actin cytoskeleton.Our preliminary studies have revealed that Rho A protein of Rho GTP family plays an important role in regulating actin cytoskeleton rearrangement,and H.pylori infection can promote the activity of Rho A protein.We also found that ROS can regulate the activity of RhoA in vitro,and H.pylori infection can stimulate the production of ROS.We hypothesized that H.pylori could enhance the invasion and metastasis of gastric cancer cells by inducing the cytoskeleton rearrangement through stimulating the production of ROS/Rho A.Objectives: In this project,we aimed to investigate the effect of H.pylori-infection on the migration,invasion,and actin cytoskeleton rearrangement in gastric cancer cells and clarify if H.pylori-infection induced ROS/RhoA is essential in these processes.Methods: 1.H.pylori(MOI 100:1)and MKN-45 cells were co-cultured to generate the H.pylori infection cell model.Phalloidin stain was used to reveal the changes of the actin cytoskeleton,and cell morphology was observed under an inverted microscope.2.The impact of H.pylori infection on cell invasion and migration was examined by transwell assay and scratch assay,respectively.3.RT-PCR was used to detect the expression level of Rho A m RNA in H.pylori-infected cells at multiple time points(0 min,15 min,30 min,60 min,180 min,and 360 min).4.GST and Western-blot were used to detect the RhoA protein activity.5.In order to clarify if Rho A is an essential molecule in the mediation of H.pylori infection-induced actin cytoskeleton rearrangement,cell invasion and migration,we used siRNA against Rho A to silence its expression,and the aforementioned impacts were assessed as above;6.H.pylori infection-induced ROS production was determined by FACS at different time points as shown above.Results: 1.MKN-45 cells become elongated and exhibited multiple protrusions after being co-cultured with H.pylori,and a loss of cell-cell contact was observed,showing a typical "hummingbird phenotype".By phalloidin staining,the elongated structures were confirmed to be actin skeleton.Cell elongation was further demonstrated by increased length to width ratio(L/B)in H.pylori infected cells(4.82±1.165,as opposed to 1.28±0.22 in PBS control group,p<0.05).2.There was a significant increase in the number of invasive cells in H.pylori-infected group as compared to control group(1600.5±109.6 491±58.6,P<0.05).By the scratch test,H.pylori infection significantly increased the migration of MKN-45 cells,as indicated by higher healing area in H.pylori infected cells(15.1±2.8%)than in the control cells(4.9±2.3%)(P<0.05).3.Increased expression of RhoA was found in the H.pylori infected cells at the protein level but not the mRNA level.4.Down-regulation of Rho A did not affect the invasion of MKN-45 cells.5.By FACS using DCFH-DA as probes,we observed a significant increase in the production of ROS in the H.pylori-infected cells together with increased RhoA protein activity.However,blocking ROS production by NAC could significantly reduce the RhoA protein activity in H.pylori infected cells.6.H.pylori infection-induced ROS production was determined by FACS at different time points as shown above.Conclusion: 1.H.pylori infection can promote the invasion and migration of the gastric epithelial cells;2.H.pylori infection can induce the rearrangement of actin cytoskeleton in gastric epithelial cells in which RhoA is an essential protein whose generation is likely medicated by H.pylori induced ROS. |
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