The Mechanism Of FOXP3 In Faciliating The Liver Metastasis In Colorectal Carcinoma

Objective: Colorectal cancer is one of the common malignant tumors in the world,and its incidence is increasing year by year.In China,the incidence of colorectal cancer ranks fourth,and the mortality rate ranks fourth.The 2018 Global Cancer Annual Report shows that the incidence of colorectal cancer is 10.2%,ranking third in the global incidence of new cancers.Distant metastasis is the main cause of treatment failure in patients with colorectal cancer.Liver metastasis is the most important target organ for blood metastasis.About 20%-34% of patients have liver metastasis at the time of clinical diagnosis.Colorectal cancer liver metastasis is also the leading cause of death in colorectal cancer patients.The mechanism of liver metastasis is still unclear.Early detection of meaningful biomarkers are the key to improving the survival of colorectal cancer patients.A series of changes related to liver metastasis of colorectal cancer are searched from the genetic level.Therefore,to explore the molecular mechanisms of the occurrence and development of colorectal cancer liver metastasis are extremely important for improving the prognosis of patients with liver metastasis of colorectal cancer patientsis and finding an effective therapeutic target for the treatment.FOXP3(Forkhead Box P3)is a transcription factor.It has been found that FOXP3 is an important marker gene of regulatory T cells(Tregs)and participates in the development and function of precisely regulated Tregs cells.However,recent studies have found that the FOXP3 gene is expressed in a variety of tumor cells.It suggests that FOXP3 may be involved in the development of tumor.At present,the function and mechanism of FOXP3 expression and expression in colorectal cancer cells are unclear,and related biological behaviors and mechanisms are still unclear.In this study,we aimed to explore the expression and function of FOXP3 in colorectal cancer tissues and liver metastasis tissues and the mechanisms involved in regulating colorectal cancer liver metastasis,and to provide new strategies for the prediction and treatment of patients with colorectal cancer liver metastasis.Methods: 1.Use immunohistochemical techniques to detect the expression of FOXP3 protein in adjacent tissues,colorectal cancer tissues and liver metastatic tissues;2.Use TCGA(The Cancer Genome Atlas)and GEO(Gene Expression Omnibus)databases and our center clinical follow-up data to analyze the relationship between FOXP3 expression and clinicopathological parameters of patients with colorectal cancer patients;3.Transfection of si RNA to establish FOXP3 transient knockdown cell lines;4.Lentiviral transfection silence and overexpression of FOXP3 to establish stable transfection cell lines;5.CCK8 experiment was used to detect cell proliferation ability;6.Flow cell technology was used to detect cell cycle changes;7.Transwell methods were used to determine cell migration and invasion ability;8.Co-immunoprecipitation method was used to detect protein binding;9.Non-target metabolic mass spectrometry was used to analyze the changes of FOXP3 affecting the small molecules of cell metabolism;10?Use luciferase experiment,PCR,Western bolt to detect the activation of Wnt signaling pathway;11.Use immunofluorescence experiment to detect the change of ?-catenin into the nucleus;12.In vivo studies were carried out using nude mouse colorectal cancer liver metastasis models;13.Statistical analysis: R software and Graphpad statistical software were used for statistical analysis.All experimental results were repeated 3 times and expressed as mean ± standard deviation(x ±s).P<0.05 was considered statistically significant.Results: 1.FOXP3 is highly expressed in colorectal cancer tissues and liver metastatic tissues.Retrospective analysis of clinical pathological data showed that high expression of FOXP3 was associated with lymph node metastasis,TNM stage,and simultaneous(or metachronous)liver metastasis.2.GEO and TCGA database analysis results show that the expression of FOXP3 in colorectal cancer is significantly related to KRAS(V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog)mutation status and microsatellite instability(MSI),while It is not related to(V-raf murine sarcoma viral oncogene homolog B1)BRAF mutation status.3.FOXP3 promotes the proliferation of colorectal cancer cells in vivo.The cell proliferation-toxicity test CCK8 suggested that the proliferation ability of tumor cells decreased after silencing FOXP3,and the proliferation ability increased after overexpressing FOXP3.4.FOXP3 promotes the invasion and migration of colorectal cancer cells.Transwell experimental results suggest that after silencing FOXP3,the migration and invasion ability of RKO and HT29 cells is reduced,and that after overexpression of FOXP3,the migration and invasion ability is enhanced.5.In vivo studies have shown that FOXP3 in nude mice promotes the proliferation of colorectal cancer cells.Lentiviral overexpression and knockdown of FOXP3 expression levels in RKO cells were injected subcutaneously in the axilla of nude mice.After 28 days,the volume of subcutaneous tumors in the axilla of the nude mice in the silent FOXP3 group was significantly smaller than that in the control group,while the tumor volume in the overexpression group was lower than that of the control group.The group grows.4.In vivo studies have shown that FOXP3 in nude mice promotes fine liver metastasis of colorectal cancer.Immunofluorescence,q PCR and Western blot fully verified the efficiency of overexpression and silencing of FOXP3 in RKO cells transfected with lentivirus.Tumor cells were injected into the tail vein of nude mice.The number of liver metastases in the over-surface group was significantly higher than that in the control group,while that in the silent group was The liver metastases in the control group decreased.8.FOXP3 promotes colorectal cancer cell epithelial cell-mesenchymal transition.The results of q PCR and Western blot experiments showed that the m RNA expression levels of E-cadherin,N-cadherin,Snail,and MMP-9decreased after silencing FOXP3,and the expression levels of E-cadherin,N-cadherin,Snail,and MMP-9 decreased after overexpression of FOXP3.rise.9.FOXP3 promotes?-catenin nucleation.Immunofluorescence experiments showed that after overexpressing FOXP3 in colorectal cancer cells,the increased ?-catenin was mainly localized in the nucleus,suggesting that FOXP3 promoted ?-catenin into the nucleus.10.The combination of FOXP3 and TCF4 directly activates the Wnt signaling pathway.GSEA analysis indicated that Wnt/?-catenin signaling pathway was significantly activated after FOXP3 increased.The dual luciferase reporter gene experiment verified that FOXP3 activates the Wnt/?-catenin signaling pathway.Co-immunoprecipitation experiments confirmed that FOXP3 directly binds to TCF4.QPCR and immunoblotting experiments showed that overexpression of FOXP3 promoted increased expression of Cyclin D1 and C-myc.Conversely,after knocking down FOXP3,the expression of Cyclin D1 and C-myc was down-regulated.11.Overexpression of FOXP3 while silencing TCF4 reverses the role of FOXP3 in promoting cell invasion and migration.The Treswell experiment results showed that the cell invasion ability increased after overexpressing FOXP3,and after silencing TCF4 with si RNA,the cell invasion ability decreased compared with the FOXP3 overexpression group.12.Overexpression of FOXP3 while silencing TCL4 reverses FOXP3 and up-regulates the expression of E-cadherin,N-cadherin,Snail,MMP-9,Cyclin D1 and C-myc.13.FOXP3 inhibits the methionine cycle(S-adenosyl methionine,SAM),reduces MMP9 promoter methylation,and promotes MMP9 expression.Non-target metabolomics analysis suggests that after FOXP3 is silenced,the inhibition of the SAM cycle is reduced,and the related metabolites are increased,suggesting that FOXP3 inhibits the SAM cycle;QPCR and Western blot experiments show that FOXP3 inhibits the SAM cycle in a concentration-dependent manner,and the SAM cycle is reduced after inhibition MMP9 promoter methylation promotes the expression of MMP9,which in turn participates in the regulation of metabolic reprogramming of colorectal cancer cells and promotes liver metastasis of colorectal cancer.Conclusion: The high expression of FOXP3 in colorectal cancer and liver metastasis plays the role of oncogenes,promoting the proliferation,invasion and migration of colorectal cancer cells;the expression of FOXP3 and colorectal cancer staging,the occurrence of simultaneous liver metastasis,KRAS mutation and MSI-H(DMMR)status-related;FOXP3 promotes epithelial-mesenchymal transition of colorectal cancer cells,promotes ?-catenin into the nucleus and directly combines with TCF4 to form a transcriptional costimulatory factor,and activates the classic Wnt/?-catenin signaling pathway to promote colorectal cancer liver Metastasis;FOXP3 promotes the expression of MMP9 by affecting the SAM metabolism of the methionine cycle,which in turn promotes the occurrence of liver metastasis of colorectal cancer.