The Role And Mechanism Of FOXO4 In The Proliferation And Metastasis Of Esophageal Squamous Cell Carcinoma Cell Lines

ObjectiveEsophageal cancer is one of the most common solid malignancies in the world and is the sixth leading cause of cancer-related mortality.Esophageal squamous cell carcinoma(ESCC)is the major histopathological type in East Asia,particularly in high-risk areas of northern China.In recent years,although ESCC has made great progress in the diagnosis and multidisciplinary treatment,its prognosis is still poor,and 5-year survival rate as low as 20-30%.The high incidence of metastasis and recurrence is still the main cause of poor prognosis,but its precise molecular mechanism is still not entirely clear.Therefore,in order to understand the relevant factors of ESCC metastasis,it is necessary to identify new prognostic biomarkers and therapeutic targets.We found that the expression of FOXO4 in esophageal cancer tissues and adjacent tissues was significantly different.The expression of FOXO4 was low in esophageal cancer tissues and was highly expressed in adjacent tissues,and the clinic pathological significance of FOXO4 expression in tissues,and its relationship with the prognosis to be further verified.In addition,studies have shown that the FOXO family plays an important role in the cell cycle,apoptosis,proliferation and metastasis of gastrointestinal cancers,and esophageal cancer has some commonality compared with other gastrointestinal tumors.Whether FOXO4 involved in the occurrence and development of esophageal cancerneed further study.The purpose of this study is to study the role and mechanism of FOXO4 in the proliferation and metastasis of esophageal squamous cell carcinoma.Through further study of the mechanism,we hope to contribute to the clinical diagnosis and prognosis of esophageal cancer.Materials and Method1.To study the role of FOXO4 in the phenotype of cell lines,and explore the expression of FOXO4 in esophageal cancer cell lines;and screen relatively low expression cell lines.2.Constructing the FOXO4 overexpressing esophageal cancer cell lines,and the effect of FOXO4 on the proliferation and metastasis of esophageal cancer cells was studied by MTT assay,plate cloning,scratching and transwell cell function experiments.3.To explore the relationship between FOXO4 expression and vimentin expression and E-cadherin expression,and to explore the relationship between FOXO4 and EMT.Result1.The results of Western blot showed that the expression of FOXO4 in TE1,Kyse30 and Caes17 cell lines was relatively low,and it was the lowest in TE1 cells.The relative expression levels of FOXO4 mRNA in experimental group and control group and NC group were 0.263 ± 0.008,0.680 ± 0.020 and 0.657 ± 0.013,respectively(P <0.05).The mRNA expression level of experimental group was significantly higher than that of NC group and control group.Western blot results showed that the expression level of FOXO4 protein in pCMV5-Flag-FOXO4 group was significantly higher than that in control group and NC group(P <0.05).The results showed that transfection of pCMV5-Flag-FOXO4 significantly up-regulated the expression of FOXO4 in TE1 and Caes17 cells.2.The growth rate of pCMV5-Flag-FOXO4 cells was significantly inhibited from 24 h,and the growth rate of experimental group cells was significantly slower than that of NC group and control group(P <0.05).But compared with NC group and control group,the difference was not statistically significant(P> 0.05).The results showed that up-regulation of FOXO4 could significantly inhibit the proliferation of esophageal cancer cells.3.After 48 hours,the sclerosis rate of scleral gap in pCMV5-Flag-FOXO4 transfection group was significantly lower than that in control group and NC group(P <0.05),while the difference of cell mobility was not statistically significant between NC group and Control group(P> 0.05).The results showed that the up-regulation of FOXO4 attenuated the migration ability of TE1 and Caes17 cells.4.Compared with the control group and the NC group,the number of transmembrane cells in the transfected group was significantly decreased(P <0.05),but there was no significant difference between the NC group and the control group(P> 0.05),This suggested that the up-regulation of FOXO4 expression reduced the ability to invasion of esophageal cancer cells.5.The number of clones in the transfection group was significantly lower than that in the control group and the NC group(P <0.05),but there was no significant difference between the NC group and the control group(P> 0.05),which indicated that the ability of clonal formation of esophageal cancer cells was weakened after up-regulation FOXO4.6.The expression of protein and mRNA in Vimentin were significantly inhibited after transfection with pCMV5-Flag-FOXO4,and the expression of E-cadherin were significantly up-regulated,and the difference was statistically significant(P <0.05),compared with control group and NC group(P <0.05).ConclusionIncreasing the expression level of FOXO4 gene could affect the proliferation ability of TE1 and Caes17 cells and reduce their migration and invasion ability.FOXO4 may play an important role as a tumor suppressor gene in the proliferation and metastasis of esophageal squamous cell carcinoma cell lines,which may play a role through the MET pathway.