Inflammation Promotes Progression Through Wnt/β-catenin Pathway Dependent Manner In Pancreatic Cancer Cells

Objective:Pancreatic cancer is one of the most fatal malignancies,with a 5-year survival rate of only<5%.However,present studies of multiple gene interaction and cellular pathways still can’t perfectly explain the initiation and development of pancreatic cancer.Previous studies had confirmed that inflammatory stimuli secreted by tumor-associated macrophages(TAM)could activate inflammation-stimulated NF-κB/PP2 Ac pathway and participate in pancreatic cancer progression.However,the molecular mechanism of the inflammatory stimuli induced pancreatic cancer progression has not been well explored.Methods:1、 The effect of macrophage-conditioned medium(MCM)on expression of genes involved in metastasis and growth regulation was evaluated by sequencing technology.2、 Gene Ontology(GO)and KEGG analyses were performed for the differentially expressed genes treated by MCM to predict the biological function and signaling pathway.3、 The model of chronic pancreatitis in nude mice was established by using the rat tail vein injection of dibutyltin dichloride(DBTC).4、 Immunohistochemistry was used to detect the expression of macrophage marker CD68,tumor related macrophages(TAMs)marker CD163 and β-catenin in protein level in the Orthotopic xenograft of nude mouse model of chronic pancreatitis.5、 The effect of FH535 on expression of genes was evaluated by microarray analyses.6、 Gene Ontology(GO)and KEGG analyses were performed for the differentially expressed genes treated by FH535 to predict the biological function and signaling pathway.7、 The effect of transformation growth factor-beta(TGF-β)on the activity of Wnt/β-catenin pathway was investigated using luciferase reporter gene assay.8、 The effect of TGF-β on the effect of gemcitabine induced apoptosis in pancreatic cancer cells was tested by flow cytometry.9、 The effect of TGF-β on the migration of pancreatic cancer cells was measured by in vitro wound-healing assay.10、 The effect of TGF-β on EMT of pancreatic cancer cells was detected by immunofluorescence assay.Results:Gene Ontology(GO)analyzed sequencing technology data.We confirmed the differentially expressed genes encode a wide range of functions,including cell communication,signal transduction,nucleoside,nucleotide and nucleic acid metabolism,etc.;KEGG analysis confirmed that these differentially expressed genes were involved in the activation of the Wnt/β-catenin signaling pathway.Via nude mouse models of orthotopic tumor xenografts and dibutyltin dichloride(DBTC)-induced chronic pancreatitis,we evaluated that inflammatory infiltration could activate the Wnt/β-catenin signaling pathway by immunohistochemistry.Then we toke the intersection from MCM treated and FH535 treated PANC-1 cells and chosed 1336 differentially expressed genes.By comparing the prognostic data of pancreatic cancer in TCGA database,34 differentially expressed genes were screened for the prognosis of pancreatic cancer.Luciferase reporter gene assay suggested that transformation growth factor(TGF-β)was the main inflammatory factors in MCM that can regulate the downstream genes through the Wnt/β-catenin signaling pathway dependent mechanism.By using q RT-PCR we confirmed that BCL2L1,BCL6,BID,BMP1,F11 R and TGM2 could be down-regulated by FH535 while being up-regulated by TGF-β among these 34 differentially expressed genes.Flow cytometry,wound-healing assay and immunofluorescence assay proved that TGF-β could promote the migration,drug resistance and EMT of pancreatic cancer through the WNT/β-catenin signaling pathway dependence mechanism.Conclusions:Taken together,our present study provides a new mechanism involved in the inflammation-driven cancer progression through Wnt/β-catenin pathway dependent downstream genes regulation.