The Molecular Mechanism Of Cancer-FOXP3 Involved In Invasion And Metastasis Of Pancreatic Cancer

BackgroundFOXP3(Forkhead Box P3)is a protein-coding gene,also known as scurfin.Diseases associated with FOXP3 include immune regulation,polyendocrinopathy and enteropathy,X-linked and insulin-dependent diabetes.The major pathways associated with FOXP3 are Th2 differentiation pathways and PI3K-mediated IL2 signaling events.Functions related to this gene mainly include transcription factor activity and sequence-specific DNA binding activity.The important paralog of this gene is FOXP4.The human FOXP3 gene contains 11 coding exons.Exon-intron boundaries are identical in the coding regions of mouse and human genes.By genomic sequencing,the FOXP3 gene is located on the p-arm of the X chromosome(specifically Xp11.23).Downregulation of FOXP3 expression has been reported in tumor specimens from breast,prostate and ovarian cancer patients,indicating that FOXP3 is a potential tumor suppressor gene.FOXP3 expression was also detected in tumor specimens derived from other cancer types,including pancreatic,melanoma,liver,bladder,thyroid,and cervical cancer.However,in these reports,the corresponding normal tissues were not analyzed,so it is unclear whether FOXP3 is an anti-tumor molecule in these tumors.In our previous experiments and study we were surprised to find that FOXP3 expression is elevated in pancreatic ductal adenocarcinomas compared to normal pancreatic tissue whereas FOXP3 expression is either missing or weakly positive in normal pancreatic tissue.Importantly,we found that patients with high FOXP3 expression had significantly higher rates of recurrence and metastasis after operation than those with low FOXP3 expression,and there was a statistically significant difference between the two groups.The high expression of FOXP3 in pancreatic cancer will promote the invasion and metastasis of pancreatic cancer cells.Therefore,we try to explore the role of FOXP3 in tumor invasion and metastasis and elucidate the underlying regulatory mechanisms based on the clinical pathological documentary level,cell level and animal model level in this project.Method1.Western blot was designed to detect the basic expression level of normal pancreatic ductal epithelial cell HPDE6C7 and various pancreatic cancer cell lines.The FOXP3 overexpression plasmid and FOXP3 sh RNA down-regulated plasmids were constructed based on the basic expression level of FOXP3.The lentiviral vector was used to stably transfect the cells to establish stable transfected cell lines.The expression of FOXP3 was detected by Western blot in these stable transfected cell lines.2.Scratch assay,transwell assay and type I collagen invasion assay were carried out using the cell lines constructed above.The effect of FOXP3 expression on cell migration and invasion was also observed by living cell workstation.Immunofluorescence F-actin staining was used to observe the effect of FOXP3 expression on F-actin distribution in cells.3.The luciferase plasmid was tranfected into both PANC-1 p LKO-Control and PANC-1 p LKO-FOXP3 cell lines,and the in situ pancreatic tumorigenicity experiment was performed in nude mice.Dynamic observed the metastasis of pancreatic cancer cells in nude mice in these two group nude mice using mouse live imaging technology.4.The constructed cell lines m RNA of PANC-1 p LV-Control and PANC-1 p LV-FOXP3 were used for RNA sequence.Combing RNA sequence data and TCGA database were used to analyze the differential genes associated with FOXP3 functional role in invasion and metastasis.The relationship between FOXP3 expression and screening differential genes was detected by RT-q PCR assay at RNA level.The TCGA database was again used to examine the relationship of FOXP3 with the most significant of the above-identified differentially expressed genes.5.And the relationship between FOXP3 and the screening differential gene was analyzed by Western blot and histochemical staining.CHIP experiment and dual luciferase assay was conducted to confirm the underlying regulation mechanism of these two genes.Result1.Basic expression level of FOXP3 in pancreatic cancer cells and the construction of stable FOXP3 cell lines.According to the basic expression levels of FOXP3 in different pancreatic ductal adenocarcinoma cell lines,FOXP3 overexpression pancreatic cancer cell lines(As PC-1 and PANC-1)and FOXP3 down-expressing cell lines(PANC-1 and MIA Pa Ca-2)were constructed.2.In vitro cell function experiments show that overexpression of FOXP3 can promote the migration and invasion of pancreatic cancer cells.Scratch experiment showed that upregulation of FOXP3 expression could promote the migration of pancreatic cancer cells.Transwell experiments indicated that upregulation of FOXP3 expression can promote the migration and invasion of pancreatic cancer cell lines,while downregulation of FOXP3 expression produced opposite effect.Type I collagen Invasion experiment and live cell workstation technology platform found that upregulation of FOXP3 expression can also promote the invasion and migration pancreatic cancer cell lines.F-actin immunofluorescence staining showed that,compared with the control group,upregulation of FOXP3 showed F-actin polarity distribution at the cell edge.The higher expression of FOXP3,the more obvious that the distribution of F-actin polarity.Taken together,the above findings suggest that FOXP3 overexpression is positively correlated with migration and invasiveness of pancreatic cancer cells.3.Mouse pancreatic tumor in situ confirmed that FOXP3 expression inhibition decrease pancreatic cancer cell invasion and migration.Compared with the control group,the mice in the interfering group showed a clear boundary between the pancreatic tumor mass and the pancreas of the mice,while the tumor cells in the control group were more obviously infiltrated into the pancreatic tissue of the mice.And distant metastasis in the interference group was also significantly less than the control group,but the size and quality of the pancreatic tumor mass in situ of the two group mice were not statistically different.4.Positive correlation between FOXP3 and MMP19 expression in pancreatic cancer cells.Combing RNA Sequence information derived from PANC-1 p LV-Control and PANC-1 p LV-FOXP3 these two stable cell lines with TCGA database information,several differential genes related to FOXP3 fuctional role of invasion and metastasis were analyzed.The relationship between FOXP3 expression and the screening differential genes at the RNA level was detected by RT-q PCR and found that the expression of FOXP3 was most closely related to the expression of MMP19.The closely correlation between m RNA levels of FOXP3 and MMP19 was also confirmed by TCGA database(r = 0.336,p <0.001).5.The expression relationship between FOXP3 and MMP19 in FOXP3 stable cell line was detected by Western blot.The positive correlation between the expression of FOXP3 and MMP19 in paraffin-embedded tissue of pancreatic cancer was confirmed by histochemical staining(r = 0.511,p <0.001).Bioinformatics analysis suggested that the potential target sequence of FOXP3 transcriptional binding existed in the promoter region of MMP19.PANC-1,a human pancreatic ductal adenocarcinoma cell line,was further selected to verify the binding site of the transcription factor FOXP3 in the promoter region of MMP19 by chromatin immunoprecipitation.And then MMP19 promoter region reporter gene vectors were conducted.Using dual luciferase reporter gene assay,we found that FOXP3 can directly bind and transactivate the expression of MMP19.And when we mutated the binding site of FOXP3 in MMP19 promoter region,the transcriptional activity was significantly inhibited,confirming that FOXP3 tumor cells can directly regulate the expression of MMP19 in pancreatic cancer cells.The CHIP assay and dual luciferase assay verified the regulatory effect of FOXP3 on MMP19.Conclusion1.The high expression of FOXP3 in pancreatic cancer cells can promote the invasion and metastasis of pancreatic cancer.2.FOXP3 can directly bind to the binding site of MMP19 promoter region,regulate the transcription and translation of MMP19,and then regulate the invasion and metastasis of pancreatic cancer.