The Inflammation Microenvironment Promotes The Progression Of Pancreatic Cancer Through PP2A/uPAR Pathway

Part ?:Prediction of PP2A downstream target genes in pancreatic cancer inflammation microenvironmentObjective:There is a close relationship between the inflammation microenvironment and the occurrence and development of tumors.Previous studies have found that the inflammation microenvironment of pancreatic cancer can inhibit the expression of PP2A,and the inhibition of PP2A can promote the occurrence and development of tumors.This section aims to predict the downstream target genes that the inflammation microenvironment inducing the downregulation of PP2A to promote pancreatic cancer progression..Methods:The PANC-1 cells were treated with MCM(establishing an inflammation microenvironment)and OA(PP2A inhibitor),and then the data of the treatment group and the control group were analyzed by sequencing and gene chip technology.The differential genes of the MCM treatment group and its control group and the differential genes of the OA treatment group and its control group were separately analyzed.Venn analysis was used to obtain the intersection between the differential genes of the two groups to find the target genes.Results:After treating PANC-1 cells with MCM,the sequencing results showed that 3316 genes were up-regulated and 2776 genes were down-regulated.After treating PANC-1 cells with OA,analyzing the gene chip results we found that there were 5161 genes Expression was up-regulated,with 5474 genes down-regulated.The intersection of the two results showed that there were 1197 genes that were up-regulated compared to the control group,and 1,253 genes that were down-regulated compared to the control group.uPAR was a gene that is differentially expressed by the two.After treatment with MCM and OA,the expression of uPAR showed a significant difference.Conclusion:uPAR may be a downstream target gene of PP2A in the pancreatic cancer inflammation microenvironment to promote the progression of pancreatic cancer.Part II:Validation of key pathway for inflammation microenvironment to promote pancreatic cancer progressionObjective:To confirm whether the inflammation microenvironment promotes the progression of pancreatic cancer by up-regulating uPAR expression through inhibiting PP2A.Methods:The virus infection technology was used to establish PANC-1 Tet-ON,PANC-1 Tet-ON PP2Ac? WT cell lines.These cells were treated with MCM to detect the uPAR expression in these cell lines.This proved whether the inflammation microenvironment regulates uPAR expression by inhibiting PP2A.The PANC-1 cell line was selected and treated with pathway inhibitors PD98059(ERK),EF24(NF-?B),SP600125(JNK),GF109203X(PKC)and then treated with OA,and then the uPAR expression level was detected to verify which kinase pathway PP2A regulates uPAR expression.Results:In PANC-1 Tet-ON cells,the cells were treated with MCM.The results showed that the up-regulation trend of uPAR expression was significantly weakened after over-expression of PP2Aca.PANC-1 cells were separately treated with PD98059,EF24,SP600125,GF109203X,and then treated with OA.The results showed in PD98059 and EF24 pathway inhibitor group the up-regulation trend of uPAR expression weakened after adding OA.Conclusion:The inflammation microenvironment can up-regulate uPAR expression by inhibiting PP2A.PP2A regulates uPAR expression through ERK and NF-?B kinase pathways,thereby promoting the progression of pancreatic cancer.Part ?:Relationship between uPAR and prognosis of patients with pancreatic cancerObjective:To analyze the expression level of uPAR in patients with pancreatic cancer and its relationship with prognosisMethods:GEPIA database was used to analyze the uPAR gene expression in patients with pancreatic cancer and adjacent cancers,the relationship between uPAR expression level and the pathological stage of pancreatic cancer and the relationship between uPAR expression level and OS and DFS in patients with pancreatic cancer.Results:In the GEPIA database,the expression of uPAR genes in pancreatic cancer samples was significantly higher than that in matched normal samples;the expression level of uPAR in cancer tissues of patients with pancreatic cancer was significantly higher than that in normal tissues;the expression level of uPAR was related to the pathological stage of pancreatic cancer;Survival analysis results showed that the OS and DFS lower expression group of the uPAR high expression group was significantly shorter and statistically significant.Conclusion:The high expression of uPAR is associated with poor prognosis in patients with pancreatic cancer.Part IV:Effects of uPAR on the proliferation,migration and adhesion of pancreatic cancer cells in vitroObjective:To investigate the effect of uPAR on the proliferation,migration and adhesion of pancreatic cancer cells in vitroMethods:A pancreatic cancer cell line PANC-1 was infected with uPAR shRNA lentivirus or negative control shRNA lentivirus,using lentivirus-infected cell technology.Real-time PCR and western blot were used to verify wether the mRNA and protein levels of uPAR were significantly reduced in uPAR knockdown cells.In the above cells,the effects of uPAR on the proliferation,migration and adhesion of pancreatic cancer cells were verified by MTT,cell scratch and plate cloning assayResults:A pancreatic cancer cell line PANC-1 was infected with uPAR shRNA lentivirus or negative control shRNA lentivirus,and stably infected cell lines were established.Real-time PCR and western blot results showed that the mRNA and protein levels of uPAR were significantly reduced in uPAR knockdown cells.The MTT and cell scratch and plate cloning assay showed that down-regulation of uPAR inhibited the cell proliferation,migration,and adhesion of pancreatic cancer cells.Conclusion:The uPAR expression level is positively correlated with the proliferation,migration and adhesion of pancreatic cancer cells in vitro.Part V:Effects of uPAR on pancreatic cancer growth and angiogenesis in vivoObjective:To investigate the effects of uPAR on the growth and angiogenesis of pancreatic cancer in vivoMethods:Subcutaneous xenograft tumor models of nude mice in the uPAR knockdown group and control group were established,and then the tumors of the experimental group were completely stripped,and the tumor weights of the uPAR knockdown group and the control group were compared.Tumor sections were immunohistochemically analyzed,and the expressions of Ki67 and CD34 in the three groups of transplanted tumors were analyzed and compared.Results:Analyzing the tumor weight of the uPAR knockdown group and the control group,it could be seen that the tumor weight of the transplanted tumor in the knockdown group was significantly smaller than that of the control group.Immunohistochemical results showed that Ki67 and CD34 expression levels were significantly lower in the uPAR knockdown group than in the control groupConclusion:uPAR expression level is positively correlated with pancreatic cancer growth and angiogenesis in vivo.Part VI:Bioinformatics analysis of differential genes after uPAR knockdownObjective:Analyze the biological function of the downstream genes of uPAR to find the downstream target genes of PP2A/uPAR.Methods:Gene chip analysis was performed on uPAR-knockdown PANC-1 cells,followed by GO analysis of differentially expressed genes.Venn analysis was used to intersect the differential genes after uPAR knockdown with differential genes after MCM and OA treatment.The STRING database and Cytoscape software were used to map protein networks of downstream genes.KOBAS and KEGG pathway analysis websites were used to perform pathway enrichment analysis on downstream genes.Results:There were abundant interactions between proteins encoded by PP2A/uPAR downstream genes.These genes were enriched in many signaling pathways.The most likely downstream target genes of this pathway were MMP1,BMP2,and IGF2.Conclusion:The most likely downstream genes for the inflammation microenvironment to promote pancreatic cancer progression through the PP2A/uPAR pathway are MMP1,BMP2,and IGF2.