| Objective: Gastric cancer is one of the major diseases that seriously affect human health.Among all malignant tumors,the death rate of gastric cancer ranks the third [1,2].Like almost all malignant tumors,the development of gastric cancer is a complex process regulated by many factors.Among them,helicobacter pylori infection is a factor that cannot be ignored.Modern epidemiological data show that Helicobacter pylori infection plays an extremely important role in the occurrence and development of gastric cancer [3].First of all,helicobacter pylori infection will lead to inflammation of the stomach.When the time of H.pylori’s persistent infection is more than ten years,it can transform superficial gastritis into atrophic gastritis.And with helicobacter pylori’s lasting effect,after a long time,the disease course will further develop,and atrophic gastritis can be transformed into intestinal metaplasia,eventually,lead to cancer [4].But not all patients infected with helicobacter pylori eventually develop stomach cancer.Therefore,the extent to which Helicobacter pylori plays a role in gastrointestinal diseases,as well as the exact pathogenic mechanism,remains to be further studied and clarified [5,6].Through database mining,this study targeted FAM60 A,a molecule with obvious changes and clinical significance in gastric cancer tissues,and conducted in-depth and systematic scientific research on the pathogenesis of helicobacter pylori by studying the interaction between helicobacter pylori and gastric cancer cells.At the molecular level,the aim is to provide new research ideas and theoretical basis for the mechanism of helicobacter pylori infection,promoting the occurrence and development of gastric cancer.Methods: 1.First,we searched public databases related to cancer,such as Oncomine,TCGA,GEO,etc.,to predict the expression of different molecules in gastric cancer and healthy gastric tissue,to select the molecular with differential expression into dataset A.The relevant survival curves of each molecule in the dataset were analyzed and plotted in the Kaplan-Meier online database,to select molecules whose variation might have clinical significance into dataset B.Again,through the search of the database,the dataset C contains molecules which expressed differently in the Helicobacter pyloriinfected gastric tissues and uninfected gastric tissues.Compared and analyzed the data set B and the data set C,we target the cross-occurring molecule FAM60 A.2.Paired pathological sections of 54 patients with gastric cancer were selected,i.e.,histopathological specimens of gastric cancer and corresponding adjacent normal histopathological specimens.We performed immunohistochemistry assay to detect FAM60 A expression levels in various histopathological specimens.We collected the patients’ clinical data,including the patient’s age,sex,tumor size,tumor stage,tumor metastasis,helicobacter pylori infection,and other factors.And we analyzed the correlation between FAM60 A expression level and the clinical data to evaluate the prognostic significance and the pathogenicity of FAM60 A in gastric cancer disease.3.Gene level and protein expression level of FAM60 A in gastric epithelial cell line GES-1 and four gastric cancer cell lines HGC-27,AGS,SGC-7901,and MGC-803,were detected by PCR and western blot assay.Two gastric cancer cell lines with a medium expression of FAM60 A were selected for subsequent experiments.The standard strain of Helicobacter pylori ATCC 43504 was cultured and cocultured with the selected two gastric cancer cell lines.PCR and western blot assay were used to detect FAM60 A m RNA and protein levels’ change of these two cell lines.Use small interfering RNA(si RNA)knock FAM60 A down in advance,then cocultured these two gastric cancer cell lines with Helicobacter pylori,respectively,and at the same time,set a blank group and a control group.CCK-8 and clone formation assay was used to detect the proliferation of gastric cancer cells;Apoptosis and cell cycle of gastric cancer cells were detected by flow cytometry;the Migration of gastric cancer cells was detected by cell scratch assay,and invasion of gastric cancer cells was detected by Tranwell assay.4.A lentivirus FAM60 A overexpression vector was designed and transfected into the two selected gastric cancer cell lines,and a blank group and a control group were set up to detect the changes in biological behaviors such as proliferation,apoptosis,cell cycle, cell migration,and cell invasion.Western blot assay was used to detect the expression changes of proteins related to apoptosis,cell cycle,cell migration,and cell invasions,such as bcl-2,bax,caspas3,cleaved caspas3,cyclin A,cyclin D,MMP9,and MMP2.5.Under the condition of different groups,such as with Helicobacter pylori infection,FAM60 A knockdown,or overexpress processing,western blot assay was used to detect the change of different pathways in the two selected gastric cancer cells.The pathway,which has an obvious change,will be inhibited by the pathway inhibitors such as LY294002(the inhibitor of the PI3K/Akt pathway).Then we detect the cell biology behavior to make sure that if the pathway has a pathological significance in the disease progression.Results: 1.Data from Oncomine et al.showed that the expression level of FAM60 A in gastric cancer tissues was higher than that in adjacent normal tissues(P<0.05).In the KaplanMeier online database,with the survival prognosis of gastric cancer patients as the background and FAM60 A as the target,the survival curve was made to show that the expression level of FAM60 A was closely related to the prognosis of gastric cancer patients,and the high level of FAM60 A was associated with the poor prognosis of gastric cancer patients(P<0.05).A sequencing study on Helicobacter pylori-infected mouse cell model in the GEO database showed that the expression level of FAM60 A in gastric mucosal cells of mice infected with Helicobacter pylori was significantly higher than that of the control group(P<0.05).2.Of the 54 clinicopathological samples,42 of them obtained complete immunohistochemical results of FAM60 A.The semi-quantitative statistical results of immunohistochemistry showed that the expression level of FAM60 A protein in gastric cancer tissues was significantly higher than that in adjacent tissues(P<0.05).Statistical analysis was conducted on the correlation between the expression level of FAM60 A in gastric cancer tissues and the clinicopathological parameters of patients.The results showed that the expression level of FAM60 A in gastric cancer tissues was significantly correlated with the tumor size,lymph node metastasis,TNM stage of the tumor,and the presence of Helicobacter pylori infection(P<0.05).3.The expression level of FAM60 A in the four gastric cancer cells was significantly higher than that in the epithelial cell line GES-1.Among the four gastric cancer cell lines,the two gastric cancer cell lines with medium FAM60 A expression were HGC-27 and AGS cells,which were selected for subsequent experiments in parallel.HGC-27 and AGS cells were co-cultured with the standard strain of Helicobacter pylori ATCC 43504,respectively,and the expression level of FAM60 A was significantly increased according to PCR and western blot detection.With the extension of coculture experiment,the increasing trend was more and more obvious.The results showed that the proliferation,migration and invasion ability of the two gastric cancer cell lines were enhanced,the apoptotic ability was weakened,and the G0/G1 phase of the cell cycle was shortened(both P<0.05).When the FAM60 A gene was knocked down in advance in the gastric cancer cells,the changes in their biological behaviors were recovered.4.After the overexpression of FAM60 A in gastric cancer cell lines HGC-27 and AGS,the proliferation,migration and invasion ability of the cells increased,the apoptotic ability decreased(all P<0.05),and the G0/G1 phase of the cell cycle shortened(P<0.05),which were the same as the results when gastric cancer cells were co-cultured with Helicobacter pylori.When PI3K/Akt pathway inhibitors are added,the affected biological behaviors are reversed.5.After co-culturing gastric cancer cell lines HGC-27 and AGS with Helicobacter pylori,western blot results showed that the expression level of p-Akt in the PI3K/Akt pathway was increased(p <0.05).When FAM60 A was knocked down in gastric cancer cells HGC-27 and AGS,and the p-Akt level was decreased(p <0.05).After overexpressing the FAM60 A gene in gastric cancer cell lines HGC-27 and AGS,the p-Akt level was increased(p <0.05),and the increased p-Akt level was reversed after the addition of the pathway inhibitor LY294002.The results of the cell biological behavior test and the nude mouse tumor-planting test in vitro also showed that the changes caused by overexpression of FAM60 A could be recovered after the addition of the pathway inhibitor LY294002.Conclusion: 1.The protein expression level of FAM60 A in gastric cancer tissues was significantly increased;The protein expression level of FAM60 A is related to the prognosis.The higher the protein expression level of FAM60 A is,the worse the prognosis of patients is.The protein expression level of FAM60 A is associated with helicobacter pylori infection,which can increase the protein expression of FAM60 A in gastric cancer tissues.2.Helicobacter pylori infection of gastric cancer cells HGC-27 and AGS can promote cell growth,cell migration,and invasion,while FAM60 A is involved in the process of Helicobacter pylori regulation of biological behavior of gastric cancer cells.3.Overexpression of FAM60 A in gastric cancer cells HGCC-27 and AGS can promote cell proliferation,inhibit apoptosis,promote cell migration and invasion,while the PI3K/Akt pathway can be activated by overexpression of FAM60 A downstream.4.The PI3K/Akt pathway is involved in the process that Helicobacter pylori promotes the high expression of FAM60 A in gastric cancer cells,thereby promoting proliferation,inhibiting apoptosis,promoting cell migration and invasion of gastric cancer cells and other changes in cell biological behavior. |
