| Backgrounds:Gastric cancer is one of the most common malignant tumor,which is originated in the gastric mucosa epithelial.Even with advanced tumor resection and molecular targeted therapy,but the prognosis of gastric cancer is very poor,the case fatality rate ranked second in tumor related diseases.While the incidence of gastric cancer in geography is different,the east of Asia,eastern Europe and South America are the high-risk areas.Causes gastric cancer has not clear,h.pylori infection,high salt diet and smoking has been confirmed that is closely related to the occurrence of gastric cancer,in which the role of helicobacter pylori infection is the most significant.Helicobacter pylori infection is associated with a variety of gastrointestinal diseases,such as asymptomatic gastritis,gastric ulcer and gastric cancer.About half of the global people had h.pylori infection,but only about 1 to 3% of the people got the cancer of the stomach,which makes the helicobacter pylori is the most common etiology of gastric cancer.Cag A is the most important virulence of h.pylori pathogenic factor,and plays an important role in the process of the pathology of gastric cancer.Cag A directly into gastric mucosal cells in h.pylori through its T4 SS system,then abnormally combined with and regulate a series of intracellular signaling pathways molecules after entering cells,causing cell morphological changes,such as cells scattered,cell elongation and cell spreading.Moreover,Cag A can lead to the reduced expression of tumor suppressor gene in cancer cells,such as P53,RUNX3,GKN1 etc,which accelerating the occurrence of cancer of the stomach and processes.Whether Cag A inhibit other tumor suppressor genes expression in gastric cancer ?KLF4 is a transcription factor containing zinc finger structure,mainly expressing in terminal differentiation of epithelial cells,such as the skin,lung,gastrointestinal tract,etc.KLF4 inhibits cell proliferation,promoting cell differentiation,and closely related with the occurrence of tumor development.In gastric cancer KLF4 play an role of tumor suppressor genes,which is significantly reduced in gastric cancer tissue compared with normal gastric mucosa cells.So,we guess whether Cag A participate in the molecular mechanism of KLF4 low expression in gastric cancer? In our previous studies have confirmed that KLF4 promoter methylation is an important cause for its reduced expression in gastric cancer.And a lot of epidemiological and clinical data confirmed that h.pylori infection can increase the cell genome methylation level,which participate in the occurrence of cancer of the stomach and process.So,whether Cag A can increase of KLF4 promoter methylation by regulating genome methylation level,resulting in its low expression in gastric cancer,finally promote the occurrence and development of gastric cancer?Therefore,in this article we first explore the relationship and molecular mechanism of Cag A and KLF4,then to detect the changes of the cell behavior,and to further explore whether the genomic methylation process involved?Research contents:1.Detection basic expression level of KLF4 in normal gastric epithelial cells and gastric cancer cell by western bloting;The high expression of KLF4 in GES-1 cells and AGS transfected Cag A plasmid,then test KLF4 expression by WB and RT-PCR and its behavioral change:(1)Detecting the proliferation ability by MTT experiment;(2)Detecting the ability to clone formation by tablet cloning experiments;(3)Detecting the ability of migration by transwell experiment.2.Establishing a stable cell line GES-1 knockdown KLF4 gene edited by CRISPR/Cas9 technique,and detected on the biological behavior change.3.Detection basic expression level of TET1 in normal gastric epithelial cells and gastric cancer cell by western bloting;gastric epithelial cell GES-1 transfected Cag A plasmid,then detected TET1 expression by WB and RT-PCR;Establishing a stable cell line GES-1 knockdown TET1 gene edited by CRISPR/Cas9 technique,and detected the expression of KLF4 by western blot.4.The lower expression of KLF4 in gastric cancer cell line transfected TET1 plasmid,then by WB and RT-PCR detected KLF4 expression and behavior change: Transwell experiment testing its ability of migration.5.To collect 88 cases of clinical gastric cancer patients and pathological specimens,then analysised the related index correlation of helicobacter pylori and gastric cancer clinical data;Immunohistochemical detected the correlation of KLF4 and 5hmc in gastric cancer tissue with the helicobacter pylori infection or not.Research results:1.KLF4 is lower expression in gastric cancer cells;2.Helicobacter pylori Cag A gene transduction results in reduced KLF4 expression in GES-1 cells and AGS cells,promoting the ability of proliferation,colony formation and migration.3.Stability on low KLF4 GES 1 cells compared with untreated cells GES-1,the ability of proliferation and clone formation and migration ability significantly enhanced; 4.TET1 is lower expression in gastric cancer cells;Helicobacter pylori Cag A gene transduction leads to reduced TET1 expression in GES-1 cells;When GES-1 cell knockdown TET1 by CRISPR/cas9 technique,the expression of KLF4 is decreased by western blot;Overexpressed TET1 in gastric cancer cells results in increased KLF4 expression,and cell migration ability also significantly reduced;5.Clinical data analysis showed that h.pylori infection was associated with lymph node metastasis of gastric cancer and tissue differentiation.Immunohistochemical results showed that expression of KLF4 and 5hmc in gastric cancer with HP infection is lower than gastric cancer without HP infection.Conclusion:1.Cag A gene transduction reduces KLF4 expression and promotes the malignant transformation of normal gastric cell GES-1 and the malignancy of gastric cancer cells.2.Cag A gene transduction reduces TET1 expression,which may associates with increased KLF4 promoter methylation.3.Expression of KLF4 and 5hmc in gastric cancer with HP infection is lower than gastric cancer without HP infection. |
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