Mechanism Of Decoy Receptor 3(DcR3) Promoting Proliferation And Invasion Of Pancreatic Cancer Cell

Objective Decoy receptor 3(DcR3)is one of the members of Decoy receptor family(DcR1,DcR2,DcR3),which belongs to the tumor necrosis factor superfamily.Studies have shown that DcR3 is highly expressed in glioma,gastric cancer,colorectal cancer,pancreatic cancer and other malignant tumors,and plays a role in inhibiting tumor cell apoptosis and promoting tumor cell immune escape,but the role and specific mechanism of DcR3 in regulating proliferation and invasion of pancreatic cancer cells have not been fully elucidated.In this study,we investigated the function of DcR3 in pancreatic cancer cells and its related regulatory mechanism.Methods(1)Immunohistochemical method(IHC)was used to detect the expression of 64 pairs of pancreatic cancer and paired non-cancerous pancreatic tissues.Serum DcR3 levels were detected by ELISA in 112 patients with pancreatic cancer.The correlation between DcR3 expression in tissue samples and serum samples was analyzed.(2)The expression of DcR3 in 5 pancreatic cancer cell lines(PATU8988,PL45,CFPAC-1,SW1990,PANC-1)was detected by reverse transcription polymerase chain reaction(RT-PCR)and Western Blot.(3)Small interfering RNA and overexpressed plasmid were used to down-regulate and up-regulate DcR3 in pancreatic cancer cell lines,respectively,and the effects of DcR3 on the proliferation and invasion ability of pancreatic cancer cells were detected by cell transwell assay,scratch assay,clone formation assay and CCK8 assay.(4)By using Gene Chip technology,the downstream differentially expressed genes in the control group and DcR3 knockout group were analyzed,and the relevant signal pathways were verified.(4)The role of the STAT1 signaling pathway in promoting the proliferation and invasion of pancreatic cancer cells by DcR3 was verified by the signal pathway functional complement experiment.(5)The transcription factor of STAT1 was found in the network database by combining the bioinformatics analysis technology with the results of gene microarray.Luciferase assay was used to verify the interaction between transcription factors and target gene promoter DNA.Results(1)The positive rate of DcR3 in pancreatic cancer tissues was 67%(43/64).The positive rate of DcR3 in non-cancerous pancreatic tissue was 28.1%(18/64).DcR3 expression in pancreatic cancer tissues was significantly higher than that in non-cancerous pancreatic tissues(χ2=19.574,P<0.001).Clinicopathological factor analysis revealed that DcR3 expression was not related to gender,age,tumor site and tumor differentiation degree(p>0.05).DcR3 expression was correlated with pancreatic cancer tumor size(P=0.040),lymph node metastasis(P=0.037),and clinical stage(P=0.010),and the difference was statistically significant.The expression of DcR3 in pancreatic cancer tissues was positively correlated with that in serum(r=0.3747,P=0.0023).The difference in survival time between the high DcR3 expression group and the low DcR3 expression group was statistically significant(log-rank,p=0.0098).(2)DcR3 was expressed highest in PATU8988 cells and lowest in PL45 cells over the 5 pancreatic cancer cell lines.Western blot was used to verify the transfection efficiency of DcR3,and the expression differences were statistically significant.Cell function assay showed that DcR3 promoted the proliferation and invasion ability of pancreatic cancer cells(P<0.05).(3)Gene microarray analysis showed that differentially expressed genes were enriched in 15 pathways,including the JAK/STAT signaling pathway(hsa04630)(P<0.05),the prolactin signaling pathway(hsa04917)(P<0.05),and the toll-like receptor signaling pathway(hsa04620)(P<0.05).Western blot results showed that DcR3 promoted the phosphorylation of STAT1,and the supplement experiment verified the role of the STAT1 signaling pathway in promoting the proliferation and invasion of pancreatic cancer cells.(4)ChIP results showed that STAT1 was bound to the P1 site of the promoter region of IRF1,but not to the P2 site of the promoter region.The double-luciferase assay further confirmed that the loss of P1 in the promoter region resulted in significantly lower luciferase activity than the wild-type vector(P<0.01),indicating that DcR3-activated STAT1 could up-regulate the expression of IRF1 by transcription.In addition,ChIP and double luciferase assay further found that IRF1 could transcriptionally regulate the expression of DcR3 and CEACAM1.DcR3/STAT1/IRF1 can increase the expression of DcR3 and promote the proliferation and invasion of pancreatic cancer cells by forming a positive feedback loop.Conclusions(1)DcR3 is highly expressed in pancreatic cancer tissues and serum of pancreatic cancer patients,and the expression levels of the two are positively correlated.The expression level of DcR3 in pancreatic cancer tissues was correlated with tumor size,lymph node metastasis and clinical staging.Survival time of pancreatic cancer patients with negative DcR3 expression was significantly higher than that of patients with positive DcR3 expression.(2)DcR3 promotes the proliferation and invasion ability of pancreatic cancer cells by up-regulating the phosphorylation level of STAT1.(3)Transcription of STAT1 upregulates the expression of IRF1 in pancreatic cancer cells,while IRF1 upregulates the expression of DcR3 and CEACAM1.Through the formation of DcR3/STAT1/IRF1 feedback loop,STAT1 is involved in the proliferation and invasion of pancreatic cancer.