| OBJECTIVES: Pancreatic ductal adenocarcinoma(PDAC)is characterized by perineural invasion(PNI).CD74 was proved to be related to PNI and overall survival(OS)in our previous study.We sought to explore the underlying molecular mechanism of CD74 in PNI.Meanswhile we explore the PNI related genes and pathways using biological analysis.METHODS: We assessed the expression of CD74 in human pancreatic cancer,pancreatic intraepithelial neoplasia(Pan IN)and chronic pancreatitis using tissue microarray,as well as in Pan INs of a Kras-mutated mouse model using immunohistochemical staining.A series of PNI assays in vivo and in vitro,including Tumor cell-Dorsal Root Ganglia(DRG)coculture assay and ectopic sciatic nerve invasion assay,were performed to elucidate the contribution of CD74 in PNI.We found the dynamic correlationship among CD74,glial cell line-derived neurotrophic factor(GDNF)and PNI in tumor-DRG coculture assay.And the correlation between CD74 and GDNFwere confirmed by ELISA,RT-PCR and tissue microarray.Biological analysis of transcription factor(TF)showed Egr-1 might regulate the expression of GDNF.It was confirmed by Chromatin immunoprecipitation(Ch IP)assays and Luciferase reporter assay.Meanswhile,GSEA showed PI3K/AKT might be the downstream of CD74.Using PI3K/AKT inhibitor LY294002 and constitutively active AKT(CA-AKT),P-AKT was proved to regulate the expression of Egr-1 and GDNF.GSE26088 and GSE40098 were chosen to analysis the PNI related genes between pancreatic cancer cell lines with high and low PNI ability.GSE102238 which included 28 pairs PNI tumor and 22 pairs non-PNI tumor was chosen to analysis the PNI related genes.Normalization and analysis of data were performed using the BRB-Array Tools sofware version v4.6.0 Beta 2.Analysis results from Gene Ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)databases were gathered and enriched by using the online Database for Annotation,Visualization and Integrated Discovery server(DAVID 6.7)with the standard enrichment computation method.PNI-related genes were introduced into Network Analyst database for protein-protein interaction(PPI)network analysis to identify the hub genes.RESULT: In Renji cohort,which included 146 PDAC patient,CD74 expression was related to PNI and independently associated with poor survival.In addition,the expression of CD74 significantly rose with histopathological progression between NP,Pan IN and PDAC.Meanswhile CD74 knockdown in PANC-1 cells suppressed PNI in vitro and in vivo.It was proved that CD74 facilitated PNI by promoting pancreatic cancer cells to secret GDNF,which can promote DRG outgrowth obviously.RT-PCR and immunohistochemical staining revealed a moderate positive correlation between CD74 and GDNF in pancreatic cancer and Pan INs.Biological analysis of transcription factor(TF)revealed the presence of a Egr-1 binding site in the GDNF gene promoter,which was confirmed by Ch IP assay and Luciferase reporter assay.GSEA showed that the PI3K/AKT signaling pathway was related to CD74 expression,and was confirmed by western blot.It was confirmed that P-AKT was the downstream of CD74 and regulated the expression of Egr-1 and GDNF.In GSE26088,we identified 445 differentially expressed genes(DEGs)related to PNI,including 176 up-regulated and 269 down-regulated genes.In GSE40098,there are 587 DEGs,including 262 up-regulated and 325 down-regulated genes,were identified.And 33 DEGs including 20 up-regulated and 13 down-regulated genes were identified.6 hub genes with degree 4 or more were identified and BDNF was reported to be related to PNI.In GSE102238,We identified 844 DEGs related to PNI,including 683 up-regulated and 161 down-regulated genes.Functional annotation showed that PNI may closely be related to “cytokine-cytokine receptor interaction”pathway.18 hub genes,with gene degree 4 or more,were identified.And four out of the top ten hub genes,including SRC,JAK2,FAS and CXCR4,were proved to be related to PNI.CONCLUSION: CD74 expression was related to PNI and was independently associated with poor survival in pancreatic cancer.It also might be an early feature of pancreatic carcinogenesis.It was proved that CD74 promote PNI mediated by GDNF.Moreover CD74 regulated GDNF expression through the PI3K/AKT/Egr-1 pathway.It might be a reliable way to find out potential PNI-related genes using human pancreatic cancer samples and cell lines,which need further work to confirm. |
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