Inflammation Promtes The Growth And Metastasis Of Pancreatic Cancer Through The Regulation Of F3 Expression By PP2Ac

Objective:Inflammation can promote the development of pancreatic cancer by activating NF-κB signaling pathway and inhibiting PP2Ac,but the molecular events involved in the downstream of PP2Ac are still unclear.Therefore,this study is to investigate the molecular mechanism of the inflammatory microenvironment to inhibit the growth and metastasis of pancreatic cancer by inhibiting PP2Ac,and it is expected to provide effective targets for gene diagnosis and gene therapy of pancreatic cancer.Methods:1.Pancreatic cancer cell PANC-1 was treated with macrophage conditioned medium(MCM)and protein phosphatase 2A(PP2A)inhibitors such as Okadaic acid(OA),Cantharidin(CAN)and interfering with PP2A lentivirus(RNAi(PP2A))respectively,using high-throughput sequencing technology of transcriptome to detect the difference of mRNA expression between MCM treatment group and control group and the microarray technique to detect the whole genome mRNA expression between OA,CAN and RNAi(PP2A)treatment group and control group.2.Construction of a cell line of PANC-1 Tet-On PP2Aca ression regulation system.The changes of F3 expression in cells was detected by real-time PCR and western blot after treatment of cells with MCM and doxycycline(DOX).3.Construction of shRNA lentiviral-mediated F3 gene interference in pancreatic cancer cell lines,which is called the PANC-1 F3-shRNA cell line.Then,do research on the effects of interference with F3 on the biological behavior of PANC-1 and related signaling pathways.4.Microarray technique was used to detect the expression of whole genome mRNA in PANC-1 F3-shRNA cells.At the same time,Funrich and Kyoto Encyclopedia of Genes and Genomes(KEGG)online software were used to analyze the function and pathway enrichment of differentially expressed genes(DEGs)downstream of PANC-1 F3-shRNA cells.5.MTT assay and plate clone formation assay was used to investigate the effect of F3 on the proliferation of pancreatic cancer cell line PANC-1;wound-healing assay in vitro was used to examine the effect of F3 on the migration of pancreatic cancer cell PANC-1;adhesion assay was used to discover the effect of F3 on the adhesion ability of pancreatic cancer cell PANC-1.6.Microarray technique was used to explore the effect of F3 on the regulation of the expression of growth and metastasis genes in pancreatic cancer cell PANC-1.7.Venn analysis was used to find out the downstream genes which can regulate growth and metastasis of pancreatic cancer after MCM inhibited PP2Ac and up-regulation of F3 expression.Results:1.The expression of F3 was up-regulated when pancreatic cancer cell PANC-1 was treated with MCM,PP2A inhibitors(OA,CAN)and RNAi(PP2A).2.In pancreatic cancer cell PANC-1,overexpression of PP2Ac significantly weakened the upregulation of F3 expression by MCM.3.Successful construction of human pancreatic cancer cell PANC-1 shRNA lentiviral-mediated F3 gene interference cell line.4.The result of whole genome expression profile chip of PANC-1 F3-shRNA showed that 869 genes were up-regulated and 1180 genes were down-regulated.The bioinformatics analysis of DEGs showed that DEGs were mainly enriched in signal transduction and cell communication in biological processes,and mainly concentrated in the nucleus and extracellular space in cell components;Molecular function is mainly enriched in transcriptional regulator activity,transcription factor activity.The results of KEGG analysis showed that DEGs mainly involved in herpes simplex virus I infection,MAPK signaling pathway,PI3K-Akt signaling pathway,TNF signaling pathway,human T-cell leukemia virus infection,EB virus infection,focal adhesion,viral carcinogenesis and transcriptional misregulation in cancer,etc.5.Interference with F3 obviously inhibited proliferation,migration and adhesion ability of pancreatic cancer cell PANC-1.6.Among the genes associated with the cell cycle,interfering F3 up-regulated CCNB1,CDKN2C,CDC20,CCNF,ID2,PPMID,CCND3,GTSE1,CCNA2,ATRIP,CDKN1A,SHC1,JAG2,and MDM2,and down-regulated GADD45A,JUN,PKD1,CCND2,and BMP2,EGFR,CDKN1C,GOS2,SESN2,TERT and PPP1R15A.7.Among the genes involved in cell motility,interfering F3 up-regulated TJP2,DIAPH1,TGFB1,CAPN1,PERP,and MST1R,and down-regulated CTNND2,MYLK,VEGFA,ICAM1,ADAMTS13,DOCK4,PVRL3,MAP1B,EGFR and RND3.8.Among the genes involved in the degradation of extracellular matrix,interfering F3 up-regulated MMP3 and down-regulated CTGF,CLEC3B,COL8A1,COL12A1,MMP11,COL16A1,COL1A1 and ADAMTS13.9.Among the genes associated with cell adhesion,interfering F3 upregulated CD44 and down-regulated CTGF,SELE,SPP1,CLEC3B,COL8A1,COL12A1,ADAMTS13,ICAM1 and COL16A1.10.Among the genes involved in epithelial-mesenchymal transition,interfering F3 up-regulated MST1R,TGFB1,SHC1,ZAK,DSC2,SYK,MMP3,TGFB3,and NOTCH4,and down-regulated COL1A1,FYN,EGFR,MAP1B,KRT19,NOTCH3,COL8A1,BPM2,COL12A1,SNAI12,ZEB2,SPP1,WNT5A and TLN2.11.MCM inhibited PP2Ac and up-regulated F3,46 genes downstream was up-regulated,and 44 genes was down-regulated.Among them,CCNB1,CCNA2,GTSE1,and GADD45A are related to growth,and MAP1B,TJP2 and CD44 are related to metastasis.Conclusion:Inflammation up-regulated expression of F3 by inhibiting PP2Ac,and then led to the up-regulation of GADD45A and MAP IB and the down-regulation of TJP2 to promote progression of pancreatic cancer.Our findings may provide a new idea for studying the molecular mechanism of the development of pancreatic cancer.