A Study Of The Role And Mechanism Of FOXP1/IRF1 Signaling Pathway In Pancreatic Cancer

Background and purpose :The continuously increasing incidence and high mortality rate of pancreatic cancer make it a major challenge in the field of public health.The mechanism of pancreatic cancer initiation and development is currently a research hotspot,and research in this field can help identify diagnostic markers and new therapeutic targets.As a transcription factor,FOXP1 can initiate transcription of different downstream molecules and play different roles in different tumors,both promoting and inhibiting cancer.Bioinformatics predictions show that FOXP1 has thousands of potential target genes,many of which play important roles in cell proliferation and apoptosis in the development of tumors.Although the role of FOXP1 in pancreatic cancer remains to be elucidated currently,abnormal cell proliferation and apoptosis plays a key role in the initiation and development of pancreatic cancer.Therefore,we hypothesize that FOXP1 plays a role in the initiation and development of pancreatic cancer,but it is unclear whether it has a tumor suppressive or tumor promoting role in pancreatic cancer,and its downstream effector molecules in pancreatic cancer are also unclear.To address these questions,we studied the expression of FOXP1 in pancreatic cancer and its association with the biological behavior of pancreatic cancer,and investigated the underlying mechanisms,in order to provide new targets for the prevention and treatment of pancreatic cancer.Materials and methods:(1)The association between FOXP1 expression and pancreatic cancer cell proliferation,growth,and prognosis in vivo and in vitro:Analysis of the association between FOXP1 and pancreatic cancer size and prognosis in clinical specimens: Immunohistochemical staining was used to detect FOXP1 expression in 72 pairs of pancreatic cancer and adjacent tissues,and the differences between them were analyzed,as well as the association between FOXP1 expression and overall survival and maximum tumor diameter of the patients.Selection of pancreatic cancer cell lines: FOXP1 protein expression levels in human normal pancreatic ductal epithelial cell line HPDE and four commonly used human pancreatic cancer cell lines CFPAC1,SW1990,PANC1,and CAPAN1 were detected by Western blotting;FOXP1 m RNA expression levels in SW1990 and PANC1 cells were compared to all other pancreatic cancer cell lines using the CCLE database.Detection of the effects of FOXP1 on pancreatic cancer cell proliferation and other biological behaviors: FOXP1 overexpression plasmids were transiently transfected into PANC1 and SW1990 cells.Cell proliferation was observed using CCK8 assay,cell cycle was detected by PI staining and flow cytometry,and cell apoptosis was detected by PI/Annexin V double staining and flow cytometry.Construction of a pancreatic cancer nude mouse model and detection of the effects of FOXP1 on tumor growth,proliferation,and apoptosis: Stable FOXP1-expressing PANC1 cell lines were constructed by transfection with pc DNA3.1(+)-FOXP1+G418 screening.A nude mouse tumor model was established by subcutaneously injecting stable transfected cell lines into the axilla.Tumor growth was evaluated by caliper measurement and tumor weight.The model was identified by HE staining and FOXP1 immunohistochemistry,and cell proliferation and apoptosis were evaluated by Ki67 immunohistochemistry and TUNEL staining.(1)FOXP1 regulates biological behaviors of pancreatic cancer cells through IRF11.Prediction and screening of potential downstream effectors of FOXP1: FOXP1 downstream effectors were predicted using TCGA,GTRD,and GSEA databases,and candidate downstream effector IRF1 was screened by q PCR.The effect of FOXP1 overexpression on IRF1 protein expression was detected by western blotting in PANC1 and SW1990 cells,and the effect of FOXP1 overexpression in vivo on IRF1 protein expression was detected by immunohistochemistry in subcutaneous tumors of nude mice.2.FOXP1 regulates biological behaviors of pancreatic cancer cells through IRF1:PANC1 and SW1990 cells were treated with FOXP1 overexpression and IRF1 knockdown rescue experiments,and cell proliferation was observed using CCK8 assay,cell cycle was detected using PI staining and flow cytometry,and apoptosis was detected using PI/Annexin V double staining and flow cytometry.3.Effect of IRF1 on CDK2 expression and biological behaviors of pancreatic cancer cells: PANC1 and SW1990 cells were transiently transfected with IRF1 overexpression plasmids,and CDK2 expression was detected by western blotting.Cell proliferation was observed using CCK8 assay,cell cycle was detected using PI staining and flow cytometry,and apoptosis was detected using PI/Annexin V double staining and flow cytometry.The protein expression level of CDK2 was compared between the FOXP1 overexpression group and the control group by immunohistochemistry in subcutaneous tumors of nude mice.(2)Identification of FOXP1 binding sites in the IRF1 promoter regionThe binding sites of FOXP1 in the IRF1 promoter region were predicted using the JASPAR website.The binding sites of FOXP1 in the IRF1 promoter region were confirmed by Ch IP-PCR and Ch IP-q PCR.The ability of wild-type and binding site mutant promoters to respond to FOXP1 and activate downstream gene transcription was detected by dual luciferase reporter gene experiments.Results:(1)The association between FOXP1 expression,pancreatic cancer cell proliferation,growth and prognosis in vitro and in vivo:1.FOXP1 is down-regulated in pancreatic cancer and affects tumor growth and prognosis: Immunohistochemical staining of 72 pairs of pancreatic cancer and adjacent tissues showed that FOXP1 expression in cancer tissues was significantly lower than that in adjacent tissues(p < 0.001).Based on the median value of the FOXP1 immunohistochemical staining score,the 72 cancer tissues were divided into a high expression group and a low expression group.The Kaplan-Meier test showed that the OS of the FOXP1 high expression group was significantly better than that of the low expression group(p < 0.001).Correlation analysis between FOXP1 expression levels and tumor size in 72 cancer tissues showed a negative correlation between the two(p < 0.001).Based on these results,we speculate that FOXP1 may be a tumor suppressor gene in pancreatic cancer.2.Selection of pancreatic cancer cell lines: Western blotting was used to detect FOXP1 protein expression levels in the human normal pancreatic duct epithelial cell line HPDE and four commonly used human pancreatic cancer cell lines CFPAC1,SW1990,PANC1 and CAPAN1.The results showed that there was a statistically significant difference in FOXP1 protein expression levels among all cell lines(p < 0.05).Comparisons between each pair of cell lines showed that FOXP1 expression levels were significantly lower in SW1990 and PANC1 cells than in other pancreatic cancer cell lines and HPDE cells(p < 0.05).CCLE database data also showed that FOXP1 expression levels were relatively low in SW1990 and PANC1 cells among all pancreatic cancer cell lines.Therefore,we decided to use these two cell lines for further experiments.3.FOXP1 affects the biological behavior of pancreatic cancer cell lines such as proliferation: FOXP1 overexpression plasmids were transiently transfected into PANC1 and SW1990 cells,and Western blotting was used to verify the overexpression effect of FOXP1(p < 0.05).FOXP1 overexpression significantly inhibited cell proliferation(p < 0.05),caused G1 phase arrest of the cell cycle(p < 0.05),and significantly promoted cell apoptosis(p < 0.05).4.FOXP1 affects the biological behavior of pancreatic cancer cells in nude mouse models: Construction of nude mouse models of pancreatic cancer and the effect of FOXP1 on tumor generation and proliferation and apoptosis: oe FOXP1/Vector-PANC1 cells were subcutaneously implanted into nude mice,and tumors were successfully formed,as confirmed by HE staining.The tumor volume and weight of the oe FOXP1-PANC1 cell group were significantly lower than those of the Vector-PANC1 cell group(p < 0.05).Immunohistochemical staining showed that FOXP1 expression in tumors formed by oe FOXP1-PANC1 cells was significantly higher than that in tumors formed by Vector-PANC1 cells(p < 0.05),while the expression of the proliferation marker Ki67 was the opposite(p < 0.05).TUNEL staining showed that apoptosis was significantly higher in tumors formed by oe FOXP1-PANC1 cells than in tumors formed by Vector-PANC1 cells(p < 0.05).These results indicate that FOXP1 can inhibit the growth and proliferation of pancreatic cancer cells and promote cell apoptosis.(2)FOXP1 affects pancreatic cancer cell proliferation and other biological behaviors by regulating the expression of IRF11.Prediction and screening of possible downstream effector molecules of FOXP1: Using TCGA,GTRD and GSEA databases,the possible downstream effector molecules of FOXP1,IRF1 and CDKN1 A,were predicted and FOXP1 overexpression significantly promoted the expression of IRF1 m RNA in PANC1 and SW1990 cells(P<0.01),but had no significant effect on the expression level of CDKN1 A m RNA(p>0.05).(p>0.05);FOXP1 overexpression significantly promoted IRF1 protein expression in PANC1 and SW1990 cells(P<0.01),and immunohistochemical staining of nude mice subcutaneous tumors showed that FOXP1 overexpression significantly promoted IRF1 protein expression in pancreatic cancer cells in vivo(P<0.01).The above results suggested that IRF1 may be a downstream effector molecule of FOXP1.2.FOXP1 affects biological behaviors such as proliferation of pancreatic cancer cells through IRF1:(1)PANC1 and SW1990 cells were treated by FOXP1 overexpression + IRF1 knockdown rescue assay,and q PCR and Western blotting assays showed that in both PANC1 and SW1990 cells,the expression of FOXP1 and IRF1 protein and m RNA was significantly higher in the oe FOXP1 group than in the corresponding Vector group(P<0.01),while the expression of FOXP1 protein and m RNA in the IRF1 knockdown group was not significantly different from its corresponding NC group regardless of whether FOXP1 was overexpressed or not(P>0.05)and the expression of IRF1 protein and m RNA in the IRF1 knockdown group was significantly lower than its corresponding NC group regardless of whether FOXP1 was overexpressed or not(P<0.01).The above results indicated that our rescue experimental model construction was successful.(2)Further study of the effect of FOXP1 regulation on IRF1 on biological behavior of pancreatic cancer showed that in both PANC1 and SW1990 cells with or without knockdown of IRF1,cell proliferation in the oe FOXP1 group was significantly lower than its corresponding Vector group(P<0.01)and apoptosis was significantly higher in the oe FOXP1 group than in the corresponding Vector group(P<0.05).The cell cycle assay also revealed that the proportion of G1-phase cells was significantly higher in the oe FOXP1 group than in the corresponding Vector group,regardless of whether IRF1 was knocked down(P<0.05).The proportion of cells in S phase was significantly higher in the oe FOXP1 group than in the corresponding Vector group(P<0.05).We also found that in both PANC1 and SW1990 cells,regardless of FOXP1 overexpression,cell proliferation was significantly higher in the IRF1 knockdown group than in the corresponding NC group(P<0.01),while apoptosis was significantly lower than in the corresponding NC group(P<0.05).and apoptosis was significantly lower than that in the corresponding NC group(P<0.05).The cell cycle assay also revealed that the proportion of G1 phase cells in the IRF1 knockdown group was significantly lower than that in the corresponding NC group,regardless of whether FOXP1 was overexpressed or not(P<0.05).The proportion of cells in S phase was significantly higher than that in the corresponding NC group(P<0.05).These phenomena were particularly evident in the oe FOXP1 group.The above results suggested that IRF1 knockdown significantly reversed the decrease in cell proliferation,cell cycle G1 phase arrest and increase in apoptosis caused by FOXP1 overexpression(P<0.05),demonstrating that FOXP1 caused the above biological effects through IRF1.3.Effect of IRF1 on CDK2 expression and cell proliferation in pancreatic cancer cells: IRF1 overexpression significantly promoted CDK2 protein expression in PANC1 and SW1990 cells(P<0.01),while significantly inhibiting cell proliferation,causing cell cycle G1 phase arrest and promoting apoptosis(P<0.01).Immunohistochemical staining of nude mice subcutaneous tumors showed that the expression level of CDK2 protein was significantly higher in the FOXP1 overexpression group than in the control group(P<0.01).(3)FOXP1 activates its transcription by binding to specific sequences in the promoter region of IRF11.The binding sites of FOXP1 in IRF1 promoter region were predicted by JASPAR website,site 1 was located at-717~-702 and site 2 was located at-552~-540;Ch IP-PCR and Ch IP-q PCR experiments in PANC1 cells showed that FOXP1 bound to both sites,but mainly to site 1(P<0.01);the double fluorophore reporter gene assay showed that the binding site 1 mutant promoter completely lost the ability to respond to FOXP1 and activate downstream gene transcription(p>0.05),and the binding site 2 mutant promoter partially lost the ability to respond to FOXP1 and activate downstream gene transcription(P<0.01),further indicating that both sites are important for FOXP1 transcriptional activation of IRF1 are important,but locus 1 is more important.Conclusions:1.FOXP1 plays an oncogenic role in pancreatic cancer and affects tumor growth and prognosis.2.FOXP1 affects the biological behaviors of pancreatic cancer,such as proliferation and apoptosis,through the regulation of IRF1.3.FOXP1 mainly binds to the-717~-702 sites and-552~-540 sites in the promoter region of IRF1,and then activates the transcription of IRF1.