Construction Of Multi-regulatory Networks In Pancreatic Cancer Microenvironment And Investigation Of Potential Therapeutic Target

BackgroundPancreatic cancer,with insipid onset and rapid progression,is the most malignant tumor in digestive tract.The overall 5-year survival rate of pancreatic cancer is still less than 10%due to its highly aggressiveness,metastatic nature and chemoresistancy.Pancreatic cancer has special tumor micro-environment(TME),in which infiltrates a large number of immune cells,while cancer cells account for less than 30%.Recent studies have shown that targeting TME can effectively improve the effectiveness of pancreatic cancer chemotherapy,which has become a research highlight at home and abroad.However,up to now,a multi-regulatory network related to the prognosis of pancreatic cancer patients has not been constructed in TME.Therefore,through big data mining and the Cancer Genome Atlas(TCGA),the establishment of a multi-regulatory network in TME can facilitate the individualized treatment of pancreatic cancer and improve the prognosis.On the other hand,it is known that tumor-associated macrophage(TAM)accounts for the largest proportion of the immune-infiltrating cells in the TME of pancreatic cancer,and plays a crucial role in the progression and chemotherapy resistance of pancreatic cancer.Combined with the preliminary research in the field of TAM by our research group,protein mass spectrometry analysis was performed on pancreatic cancer cells stimulated by gemcitabine chemotherapy and pancreatic cancer cells co-cultured with M2 type TAMs.The results showed that the pancreatic cancer cells may secrete interferon-stimulating gene of 20 kDa(ISG20)through the stimulation of gemcitabine and TAMs in TME,thus promoting the development of pancreatic cancer.At present,ISG20 has been reported to play an important role in a variety of tumors and immune responses,but no research work was found in the field of pancreatic cancer.Therefore,in addition to the comprehensive bioinformatics analysis of the TME and prognosis of pancreatic cancer,this thesis also investigated the relationship between the known protein ISG20 and the biological behavior of pancreatic cancer cells and its clinical significance.ObjectiveThe aim of this study is to construct a prognostic multi-regulatory network in the pancreatic cancer TME and explore valuable regulatory molecules for the prognosis of pancreatic cancer patients.To explore the regulatory effects of ISG20,a potential therapeutic target in TME,on malignant biological phenotypes of pancreatic cancer,as well as its downstream regulatory signaling pathways.And to clarify the expression and prognostic value of ISG20 in pancreatic cancer tissues.MethodsⅠ.Construction of Multi-regulatory Networks in Pancreatic Cancer MicroenvironmentKaplan-Meier analysis was used for survival analysis of pancreatic cancer patients.Differential analysis was performed on the downloaded data using Limma package in R,with differentially expressed genes(DEGs)being obtained through screening of significance.KOBAS database was used to perform pathway enrichment analysis on DEGs.STRING database was used to construct a protein-protein interaction network.Cytoscape software was used to screen and mine the key genes in the gene network.Ⅱ.The Role and Mechanism of ISG20 as a Potential Therapeutic Target in Pancreatic Cancer Tumor Immune Microenvironment1.Investigation of biological behaviors of pancreatic cancer cell by cell proliferation assay,Transwell assay and cytotoxicity assay.2.RNA-seq was used to help screen the target of ISG20.3.Immunohistochemical staining was used to evaluate the relationship between ISG20 expression and clinicopathological features of pancreatic cancer patients as well as its prognostic value.ResultsⅠ.Construction of Multi-regulatory Networks in Pancreatic Cancer Microenvironment1.In the pancreatic cancer cohort of the TCGA database,the cases at Stage Ⅱ subgroup had the highest average immune score,followed by Stage Ⅲ and Ⅳ,while the Stage Ⅰsamples had the lowest immune score.2.Pancreatic cancer patients with low immune scores had significantly longer 5-year survival compared with those with high immune scores.3.Genes related to the prognosis of pancreatic cancer and other tumors obtained from the module include:CXCL5,CXCL9,CXCL10,CXCL11,PPY,LPAR3,HCAR3,ANXA1,SSTR1,SSTR5,INSL5,CCL28,KISSIR and P2RY2.4.The ncRNA-pivot nodes with significant regulatory effects on prognostic genes of pancreatic cancer include:WSPAR,hsa-miR-4425,hsa-miR-4536-5p,hsa-miR-410-5p,hsa-miR-8064,hsa-miR-6871-5p,hsa-miR-4767,hsa-miR-608,hsa-miR-4481,hsa-miR-4481 and hsa-miR-3605-3p.5.The TF-pivot nodes with significant regulatory effects on pancreatic cancer prognostic genes include:RFWD2,GATA6,CITED2,PER2,TGIF1,RARA and NR5A1.6.The drug-pivot nodes with potential therapeutic effects for pancreatic cancer include:Fibrinolysin,Urokinase,Pasireotide,Octreotide,Lutetium Lu 177 dotatate,Tenecteplase,Amcinonide,Botulinum Toxin Type A,HSPE7 and Lanreotide.Ⅱ.The Role and Mechanism of ISG20 as a Potential Therapeutic Target in Pancreatic Cancer Tumor Immune Microenvironment1.Regulation of malignant biological behavior of pancreatic cancer by ISG20ISG20 significantly promoted the invasion and metastasis ability of pancreatic cancer cells in vitro and in vivo,and reduced the sensitivity of pancreatic cancer cells to gemcitabine chemotherapy,but did not affect the proliferation ability of pancreatic cancer cells in vitro.2.Molecular mechanism of ISG20 in the regulation of malignant biological behavior of pancreatic cancerThe integrin alpha 3(ITGA3)was screened.After transient knockdown,the integrin beta 1(ITGB1),c-Jun N-terminal kinase,and c-Jun N-terminal kinase(JNK)were found.The tyrosine phosphorylation level at site 185 were significantly decreased,while the expression level of equilibrative nucleoside transporter 1(ENT1)was significantly increased.3.Prognostic value of ISG20 in pancreatic cancer tissuesThe expression level of ISG20 was related to the prognosis of patients.The overall survival time of patients with high ISG20 expression was significantly shorter than that of patients with low ISG20 expression.ConclusionsⅠ.Construction of Multi-regulatory Networks in Pancreatic Cancer Microenvironment1.A network of core prognostic factors related to pancreatic cancer immune microenvironment was constructed,including DEGs related to pancreatic cancer prognosis,non-coding RNAs with significant regulatory effects on prognostic genes,transcription factors,and drugs with potential therapeutic effects.2.The prognostic related factors based on big data mining in this study not only play an important role in the occurrence and development of pancreatic cancer,but also contribute to the improvement of pancreatic cancer treatment,evaluation of patient prognosis,early diagnosis and individualized treatment.Ⅱ.The Role and Mechanism of ISG20 as a Potential Therapeutic Target in Pancreatic Cancer Tumor Immune Microenvironment1.ISG20 could significantly promote invasion and metastasis of pancreatic cancer cells in vivo and in vitro,as well as chemoresistancy to gemcitabine.2.ISG20 plays a role by activating α3β1-JNK-ENT1.3.The high expression of ISG20 indicates a poor prognosis and can be used as a potential therapeutic target.