| China is a country with a high incidence of gastric cancer.The diagnosis and treatment of early gastric cancer is effective.The 10-year survival rate is nearly 90%.However,the detection rate of early gastric cancer is still low.More than 60%of patients have entered the middle and advanced stage at the first visit,and their 5-year overall survival rate is less than 30%.Early gastric cancer is confined to the mucosa and submucosa,lacking specific symptoms,so the early detection rate is not high.When the symptoms of dyspepsia,anemia and other symptoms are consulted,the condition has often developed to the middle and late stage.At this time,the effect of surgical treatment alone is poor,requiring combined chemotherapy,biological treatment and other means.The occurrence of gastric cancer is accompanied by abnormal expression of many genes,including over-expression of proto-oncogene and down-regulation of anti-oncogene expression or loss of function.In recent years,people expect to find new methods to treat gastric cancer at the molecular level,and find specific molecular markers for early diagnosis and molecular targeted therapy of gastric cancer.Forkhead box A2(FOXA2),also known as hepatocyte nuclear factor 3 beta(HNF3 beta),is an important transcription factor.Its role in the process of tumorigenesis has attracted wide attention.The down-regulation or loss of FOXA2 expression is closely related to the occurrence and development of hepatocellular carcinoma,gastric cancer and lung cancer.Overexpression of FOXA2 can significantly inhibit cell proliferation,promote cell apoptosis and reduce the ability of cloning.It indicates that FOXA2,as a recognized tumor suppressor,may have potential function as a target of molecular targeted therapy,but its specific mechanism in gastric cancer is still unclear.Objective:Recombinant adenovirus carrying FOXA2 gene was constructed and acquired.To investigate the effect of adenovirus-mediated over-expression of FOXA2 on the growth of gastric cancer cells and its mechanism.Method:According to the sequence of FOXA2 gene,specific primers were designed and synthesized to amplify FOXA2 gene.The full-length coding gene of FOXA2 was obtained by PCR.The recombinant adenovirus expression vector was constructed.After repeated infection of HEK293 cells and virus amplification,the recombinant adenovirus Ad5.FOXA2 was purified by cesium chloride density gradient centrifugation,and the stable and highly effective recombinant adenovirus Ad5.FOXA2 was further obtained.The recombinant adenovirus Ad5.FOXA2 was used to infect gastric cancer cell line AGS.The transcription and protein expression of FOXA2 gene were detected by real-time fluorescence quantitative PCR and Western blot.Methyl thiazolyl tetrazole(MTT)was used to detect the effect of Ad5 FOXA2 on the proliferation of gastric cancer cells.Western blot was used to detect the specific molecular mechanism of Ad5-FOXA2 on the proliferation of gastric cancer cells.The effects of Ad5.FOXA2 on the migration of gastric cancer cells were examined by scratch test and cell invasion test.Results:The recombinant adenovirus Ad5.FOXA2 carrying FOXA2 gene was successfully constructed and purified.The titer of the recombinant adenovirus was 2.12×1015 pfu/L.The transcriptional levels of FOXA2 gene in gastric cancer cells infected with adenovirus Ad5.FOXA2 were 56.72±2.73 and 1.01±0.112.At the protein level,FOXA2 gene expression was 3.85 times higher than that of the control group,which was significantly up-regulated(P<0.001,P<0.001).MTT assay showed that with the prolongation of time,Ad5.FOXA2 group could significantly inhibit cell proliferation compared with the cell control group,and the difference was statistically significant(P<0.05).Wound healing showed that the migration rates of Ad5.FOXA2 group and control group were 52.62%±4.86%and 98.86%±6.59,respectively,with statistical significance(F=78.94,P<0.001).Transwell showed the migration rates of Ad5.FOXA2 group and control group were 20.79±4.06 and 50.19±4.93,respectively.The difference was statistically significant(F=118.0,P<0.001).Western blot showed that the expression levels of c-Myc,-catenin,Cyclin D1 and PCNA were down-regulated by 32%,45%,42%and 36%respectively after the expression of exogenous FOXA2(P<0.05).Conclusions:Recombinant adenovirus Ad5.FOXA2 can be effectively expressed in gastric cancer cell line AGS,and exogenous FOXA2 expression can inhibit the proliferation and migration of gastric cancer cells,which may be achieved by inhibiting the expression of c-myc,beta-catenin and Cyclin D1 proteins. |
