| Objective: Pancreatic cancer is currently one of the solid tumors with extremely high malignancy,poor response to radiotherapy and chemotherapy,and a survival rate of less than 10%.Due to its hidden anatomical location and inconspicuous symptoms,early diagnosis of pancreatic cancer patients is difficult,surgical resection rate is low,and distant metastasis is easy,the prognosis is extremely poor.FOXO3,a member of the Fox O family,is a transcription factor that mediates multiple physiological and pathological processes by inducing genes involved in transcription of cell proliferation,apoptosis,cell cycle progression,cell survival,and DNA damage.FOXO3 is also involved in tumorigenesis,development and malignant transformation.Dysregulation of FOXO3 will cause abnormal regulation of pro-apoptotic factors and lead to changes in the biological behavior of tumor cells.Moreover,FOXO3 may play diametrically opposite roles in different types of tumors.In order to clarify the relationship between FOXO3 and pancreatic cancer and its effect on pancreatic cancer cells and related mechanisms,this study mainly used database combined with clinical sample validation to obtain the expression of FOXO3 in pancreatic cancer,and conducted cell experiments to elucidate the effect of different FOXO3 expression on the biological behavior of pancreatic cancer cells,so as to provide new methods and strategies for the comprehensive treatment of pancreatic cancer patients.Methods: The expression dataset of FOXO3 in pan-cancer was obtained using GEPIA(Gene Expression Profiling Interactive Analysis)database to study its differential expression in pancreatic cancer tissues and normal pancreas,and to obtain the survival analysis results of patients with different FOXO3 expression by Kaplan-Meier Plotter survival analysis tool,then combined with the clinical information analysis of pancreatic cancer patients to verify the expression of FOXO3 and its effect on the prognosis of patients,also to detect the subcellular localization of FOXO3 in pancreatic cancer cells by immunofluorescence assay.QRT-PCR and Western blot experiments were performed to detect the m RNA and protein expression of FOXO3 in pancreatic stellate cell HPSC and four pancreatic cell lines ASPC-1,Capan-1,PANC-1,and Mia Paca-2,then two cell lines with lower expression wereselected for subsequent experiments.The FOXO3 gene sequence was constructed and inserted into the p IRES2-EGFP plasmid vector,and the plasmid was transfected into the above two cell lines,then transfection efficiency in stably transfected cells were detected through q RT-PCR and Western blot.Aim to elucidate the function of FOXO3 in pancreatic cancer cells,the changes of proliferation,migration,invasion and cell cycle of pancreatic cancer cells after G418 selection were tested by colony formation assay,CCK-8 assay,scratch assay,transwell assay and cell cycle assay respectively.Results:According to the database information,the expression trend of FOXO3 was not consistent in different tumors,and the expression of FOXO3 was higher in pancreatic cancer tissues than in normal pancreatic.FOXO3 was highly expressed in28(45.9%)pancreatic cancer tissues,but only 1(7.7%)in normal pancreatic.FOXO3 expression was significantly elevated in pancreatic cancer patients compared to normal tissues(p<0.001).The expression of FOXO3 was significantly different from both the primary site of the tumor(p<0.001)and differentiation of pancreatic cancer patients(p=0.005),but not from the clinical stage of the tumor(p<0.05).The OS of patients in the low expression group was significantly shorter compared with patients with high FOXO3 expression in tumor tissues(p<0.05).Immunofluorescence experiments revealed that FOXO3 was mainly located in the nucleus of pancreatic cancer cells,and a small proportion was scattered in the cytoplasm.Pancreatic cancer cells overexpressing FOXO3 and negative control were obtained by transfection of PANC-1 and Mia Paca-2 with plasmid vectors.The biological behavior of pancreatic cancer cells was continued after q PCR and western blot to detect the efficiency of cell transfection.Both colony formation assay and CCK8 assay showed that the cell proliferation ability of pancreatic cancer cells was reduced after overexpression of FOXO3,and the difference was statistically significant(p<0.05).Scratch assay and Transwell assay results showed that the migration and invasion of pancreatic cancer cells were significantly reduced after upregulation of FOXO3(p<0.05).The results of cell cycle experiments suggested that the proportion of pancreatic cancer cells in G0/G1 phase decreased and the proportion in S phase increased after upregulation of FOXO3 expression compared with negative control,and the difference was statistically significant(p<0.05).Conclusions:The expression of FOXO3 in pancreatic cancer is higher than that in normal tissues,and FOXO3 is related to the differentiation and prognosis of pancreatic cancer.High FOXO3 expression may be a predictor of better tumor differentiation and prognosis in patients with pancreatic cancer.In vitro,upregulation of FOXO3 expression could significantly inhibit the proliferation,migration and invasion of pancreatic cancer cells,and FOXO3 can inhibit the proliferative activity of pancreatic cancer cells through S phase arrest in the cell cycle. |
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