Helicobacter Pylori Promote B7-H1 Expression By Suppressing MicroRNA-152 And MicroRNA-200b In Gastric Cancer Cells

Part I Helicobacter Pylori Promote B7-H1 Expression by Suppressing miR-152 and miR-200b in Gastric Cancer CellsAims:By analyzing the B7-H1 expression of tumor tissues of gastric cancer patients and the influence of Helicobacter pylori on B7-H1 expression in gastric cancer cells,this study aims to investigate the mechanism of how Helicobacter pylori regulate the B7-H1 expression in gastric cancer cells,suggesting potential targets for the prevention and treatment of gastric cancer.Methods:Gastric cancer tissue samples were collected from gastric cancer tissue removed from patients by surgery.We tested the B7-H1 expression levels of the samples and analyzed the correlation between the B7-H1 positive rate and the factors of gender,age,differentiation level,TNM stage and Helicobacter pylori infection;samples were defined as B7-H1-positive if the ratio of B7-H1-positive cells to Epcam-positive cells in the specimen was>10%.We cultured the human gastric carcinoma(AGS)cells in 6-well plates and Helicobacter pylori were routinely grown on tryptic soy agar plates supplemented with 5%sheep blood in mixed air containing 10%CO2,5%O2 and 8%N2 at 37?.After a 24 hours co-culture of AGS cells and Helicobacter pylori(HP/cell ratio of 10/1),the expression level of B7-H1 were detected by flow cytometry.The microRNA profiles of B7-H1 positive AGS cells and B7-H1 negative cells were analyzed by microRNA array.The candidate microRNAs were selected by the prediction software,the candidate microRNAs mimics were transfected into AGS cells,the corresponding B7-H1 expression levels were detected by flow cytometry.Luciferase reporter vectors were designed to identify microRNAs targeting the mRNA of B7-H1.The microRNAs levels of B7-H1 positive AGS cells and B7-H1 negative cells were detected by qRT-PCR.By qPCR and flow cytometry,we examined the correlation between the microRNAs levels and the B7-H1 expression levels in gastric cancer tumor tissues with Helicobacter pylori infection.Results:The B7-H1 positive rate of gastric cancer tissue collected from male patients was 53.6%,and from female patients it was 65.0%;the B7-H1 positive rate of gastric cancer tissue collected from aged patients(>60)was 50.0%,and from non-aged(<60)patients it was 61.4%;the B7-H1 positive rate of high differentiation gastric cancer tissue was 55.6%,and from low differentiation gastric cancer tissue it was 59.6%;according to TNM stages,the B7-H1 positive rate of gastric cancer tissue collected from stage I patients was 37.5%,from stage ? it was 47.3%,from stage III it was 56.4%and from stage IV it was 73.3%;the B7-H1 positive rate of gastric cancer tissue with Helicobacter pylori infection was 64.5%,and without Helicobacter infection it was 35.0%.We found a marked increase in B7-H1 expression on the surface of the AGS cells co-cultured with Helicobacter pylori for 24 hours and the IFN-y treatment also markedly stimulated B7-H1 expression in AGS cells,Furthermore,Helicobacter pylori and IFN-? had an additive effect on stimulating surface B7-H1 expression in the AGS cells.By microRNA array screening and prediction software,we selected 5 candidate microRNAs;after transfecting the candidate microRNAs and corresponding inhibitors into the AGS cells,we found that microRNA-152 and microRNA-200b had an obvious down regulation influence on B7-H1 expression level in gastric cancer cells.By luciferase report system,we found that the microRNA-152 and microRNA-200b could bind to the 3'UTR of B7-H1 mRNA.In gastric cancer tissues with Helicobacter pylori infection,we found a negative correlation between B7-H1 expression levels and the two microRNAs levels in gastric cancer cells.Conclusions:Helicobacter Pylori Promote B7-H1 Expression by Suppressing miR-152 and miR-200b in Gastric Cancer Cells.Part ? Promoter Hypermethylation of microRNA-152 and microRNA-200b Increased B7-H1 Expression by Reducing microRNAs Expression in Gastric Cancer CellsAims:To investigate the mechanism of how the microRNAs expression regulates B7-H1 expression in gastric cancer cells.Methods:Treat the human gastric cancer cell lines with 5-Aza and test the B7-H1 expression after treatment via FACS.The expression of microRNA-152 and microRNA-200b were tested by qRT-PCT after treatment with 5-Aza.AGS cells were transfected with the specific inhibitors of the two microRNAs,then the transfected cells were treated with 5-Aza,the expression of B7-H1 were examined via FACS.Luciferase reported assay was operated for verifying the effect of 5-Aza on the microRNAs expression in AGS cells.The correlations between the promoter hypermethylation level and the microRNAs expression level or the B7-H1 expression level were analyzed in gastric cancer tumor samples.Results:Hypermethylation of microRNA promoters resulted in the down-regulation of microRNA expression and the up-regulation of B7-H1 expression in gastric cancer cells.Conclusions:Helicobacter Pylori Promote B7-H1 Expression by Suppressing miR-152 and miR-200b in Gastric Cancer Cells.