The Role Of GCN2 On Helicobacter Pylori-induced Gastric Carcinoma And Its Relationship With Autophagy

Background:Helicobacter pylori(H.Pylori),a class I carcinogen identified by the World Health Organization,plays an important role in gastric carcinoma.The importance of autophagy in the development of tumors has received increasing attention,but its mechanism in Helicobacter pylori infection-induced gastric cancer is not clear.General control nonderepressible-2(GCN2)is a key regulator of protein metabolism which participates in a variety of cell activities such as stress,metabolism,autophagy,and inflammatory reaction.GCN2 has been preliminarily confirmed to be involved in the process of carcinoma,such as lung cancer,colon cancer and breast cancer.However,few studies have been conducted on the role of GCN2 on Helicobacter pylori infection-induced gastric cancer.Therefore,it is the focus of this research whether autophagy is involved in Helicobacter pylori infection-induced gastric cancer through the regulation of GCN2 on Beclin1(BECN1),which is an important regulator of autophagy for the formation and maturation of autophagosomes.Objective1.To investigate the function of GCN2 in the process of gastric carcinoma.2.To explore the role of GCN2 in gastric cancer cells autophagy and the relative regulatory mechanism.Methods1.CeHular and molecular levelsThe level of GCN2 expression in four gastric mucosal epithelial-derived cell lines(GES-1,AGS,BGC-823 and SGC-7901)was detected by Western blot and qRT-PCR.With Helicobacter pylori infection or LPS stimulation,it was examined the expression changes of GCN2 and BECN1 by Western blot.It was also checked whether LPS could affect gastric cancer cell autophagy through GCN2 by the recovery experiment.The formation of autophagosomes was observed through transmission electron microscopy.After transfected with GCN2 small interfering RNA,the expression of BECN1 was verified by Western blot.Furthermore,the effect of GCN2 on the proliferation of gastric cancer cells was decided by Clone genetics assay.The role of ATF4 on the expression BECN1 induced by GCN2 was also detected in this process.2.Animal models levelThe subcutaneous tumorigenesis test in nude mice was performed to observe the tumor growth and the tumor size.Western blot,qRT-PCR,immunohistochemistry and transmission electron microscopy were used to detect the role of GCN2 in the development of gastric cancer and the relative mechanism of autophagy..3.Pathological specimens levels of human gastric cancer tissueThe expression of GCN2 and BECN1 in 12 pairs of human gastric cancer tissues and adjacent normal tissue samples was detected by Immunohistochemistry assay.The correlations between the expression levels of GCN2 and BECN1 in gastric carcinoma and their relationship with the disease prognosis were also further analyzed in combination with bioinformatics data.Results1.Cellular and molecular levelsGCN2 was stably expressed in four gastric epithelial cell-derived cell lines.After the infection of Helicobacter pylori or the stimulation of LPS,the expressions of GCN2 and BECN1 were both elevated in the cells,which was positive-related with the time and the concentration of the stimulation.It was confirmed by the recovery experiment that LPS could affect gastric cancer cell autophagy and the formation of autophagosomes through regulating the expression of GCN2.After the inhibition of GCN2 expression,it was suggested that BECN1 and ATF4 expression decreased with the lower level of GCN2 expression,which inhibited the proliferation of gastric cancer cells.GCN2 also regulated the expression of BECN1 through ATF4-related pathway and thus affected the autophagy of gastric cancer cells.2.Animal models levelTumor growth and tumor size were both inhibited with the knockout of the GCN2 gene in the gastric cancer cells.Autophagy was also significantly inhibited with the decreased number of the autophagosomes.3.Pathological specimens levels of human gastric cancer tissueCombined with immunohistochemistry detection and bioinformatics data analysis,the expression level of GCN2 in human gastric cancer tissue specimens was significantly higher than that in the adjacent ones.The autophagy-associated protein BECN1 was also highly expressed in gastric cancer species which had a positive correlation with GCN2 expression.The expression of GCN2 was associated with the prognosis of gastric cancer patients.ConclusionGCN2 is overexpressed in human gastric cancer species and participates in gastric carcinoma.Helicobacter pylori regulate the expression of autophagy-related proteins through GCN2,which promotes autophagy and gastric carcinoma.This study has preliminarily explored the importance of GCN2 in H.pylori-induced gastric cancer and the related molecular mechanism that can provide a new theoretical basis for the early pre-vention and treatment of gastric cancer.