Effect Of Pancreatic Stellate Cells Derived Exosomal Mir-21 On Migration Ability Of Pancreatic Cancer Cells And Study Of The Prognostic Marker SERP1 Of Pancreatic Cancer

Pancreatic ductal adenocarcinoma(PDAC)is a common malignant tumor of human digestive system.Its symptoms are hidden,it is difficult to diagnose early and the mortality rate is high.Most patients have progressed to the advanced stage when they are diagnosed.As a special type of cancer-associated fibroblast(CAF),pancreatic stellate cells(PSCs),aberrantly activated in PDAC tissues,can promote the proliferation,invasion and migration of PDAC cells and induce drug resistance.However,the study of the effect of PSCs on its malignant biological behavior is limited.More and more studies have found that CAFs could regulate tumor cell biological behavior by releasing exosomal miRNA.It was reported that miR-21 was significantly up-regulated in the activated PSCs derived exosomes in PDAC,and we found that high miR-21 level was closely related to lymph node metastasis,tumor recurrence and shorter survival in PDAC patients by The Cancer Genome Atlas(TCGA)database analysis.However,the potential mechanism of PSCs derived exosomal miR-21 regulating PDAC biological behavior was still unclear.Therefore,in the first part,we investigated the effect and mechanism of PSCs-derived exosomal miR-21 on migration of PDAC cells at cellular level.In addition,in order to find novel prognostic markers for PDAC patients,the second part of this study mainly focus on the potential value of stress associated endoplasmic reticulum protein 1(SERP1)in prognosis of PDAC patients.The purpose of this study is to provide a preliminary theoretical basis for the study of PSCs-derived exosomal miR-21 mediated PDAC cells migration and to find potential prognostic markers for PDAC patients.Part ? The effect and mechanism of pancreatic stellate cells derived exosomal miR-21 on migration of PDAC cells1.Previous study found that miR-21 was significantly up-regulated in activated PSCs derived exosomes,hence,we further analyzed the role of miR-21 in progression of PDAC by bioinformatics methods.The results showed that patients with high miR-21 levels had higher T and N stages,and shorter overall survival(OS)and disease-free survival(DFS),indicating miR-21 may be a key factor in the occurrence and development of PDAC.KEGG analysis confirmed that target genes of miR-21 mainly involved in regulating Ras and Erk signaling pathways.And the frequency of gene mutation of PDAC patients was analyzed by TCGA database,and the mutation rate of K-ras was 91%and Ras signaling pathway was over-activated in PDAC patients.Therefore,bioinformatics analysis suggested that PSCs derived exosomal miR21 may promote the progression of PDAC by regulating Ras signaling pathway.2.In order to further verify the relative exosomal miR-21 levels in PSCs and PDAC cells,we firstly cultured primary PSCs by freshly resected pancreatic cancer tissue and then isolated exosomes from PSCs culture medium by ultracentrifugation.Electron microscopy and western blot were used to identify the exosomes.qRT-PCR results showed that compared with PDAC cells and deactivated PSCs,activated PSCs derived exosomes had higher miR-21 levels.3.In order to observe whether PSCs derived exosomal miR-21 could be uptaken by PDAC cells,in vitro,PSCs derived exosomes were labeled by PKH67 green fluorescent dyes,then were added to culture medium of PANC-1 and MIAPaCa-2 cells.After 3 hours,fluorescence microscopy observed that PSCs derived exosomes with green fluorescence were uptaken by PDAC cells.Next,qRT-PCR further analyzed the relative miR-21 levels in the PDAC cells treated by exosomes or not,and found that the relative miR-21 level was significantly up-regulated in those PDAC cells treated by exosomes compared with untreated cells.The results showed that PSCs derived exosomal miR-21 could be absorbed by PDAC cells.4.To observe the effect of PSCs derived exosomal miR-21 on the malignant phenotype of PDAC cells,PANC-1 and MIAPaCa-2 cells were treated with PSCs derived exosomes.The results showed PSCs-derived exosomes could significantly enhance the migration abilities of PANC-1 and MIAPaCa-2 cells.Western blot and gelatin zymography results showed that after PDACs were treated with PSCs derived exosomes,the expression level of epithelial-mesenchymal transition(EMT)-related markers was changed,while the matrix metalloproteinases 2 and 9(Matrix metalloprotein 2 and 9,MMP-2/9)activity was increased.In order to determine whether the promoting migration ability of PSCs derived exosomes was mediated by miR-21,miR-21 inhibitor was transfected into activated PSCs to suppress miR-21 endogenous expression,then their exosomes were collected.Down-regulation of PSCs derived exosomal miR-21 could reverse PSCs exosomes-mediated cell migration and EMT,and also reduce MMP-2/9 activity in PDAC cells.5.According to bioinformatics analysis results,western blot confirmed that PSCs derived exosomal miR-21 could up-regulate the expression level of Ras,phosphorylation-Erk and phosphorylation-Akt in PDAC cells.Protein-protein interaction(PPI)analysis revealed that RAS p21 protein activator 1(RASA1)is an important gene,and it was not only involved in Ras and Erk signaling pathways but also is a key target gene of miR-21.Moreover,it is also a negative regulator of Ras signaling pathway.Western blot results further confirmed that PSCs derived exosomal miR-21 could promote Ras/Erk/Akt pathway activation by downregulating RASA1 expression in PDAC cells.6.Transcription factor activation profiling plate array found that the activity of signal transducer and activator of transcription 3(STAT3)was 10 times higher in activated PSCs than that of ATRA-induced PSCs.Western blot was further used to detect the expression level of phosphorylated and non-phosphorylated STAT3 in PSCs from three PDAC patients.The results showed that the expression level of phosphorylated STAT3 was significantly higher in activated PSCs than that in ATRA-induced PSCs.Because STAT3 is a transcription factor of miR-21,the relative level of endogenous miR-21 and exosomal miR-21 were both down-regulated after transfecting si-STAT3 into activated PSCs.Part ? The relationship between SERP1 and prognosis in pancreatic ductal adenocarcinoma patients and its role in regulating tumor cell apoptosis1.GEO database(Gene Expression Omnibus,GEO)was used to download microarray data of PDAC patients,and R language was used to analyze differentially expressed mRNA.The relative mRNA level of SERP1 was significantly higher in PDAC tissues compared with adjacent tissues.In Human Protein Atlas(HPA)database,immunohistochemical data also confirmed that the expression level of SERP1 in PDAC tissues was also significantly up-regulated compared with adjacent non-cancerous tissues.Immunohistochemical analysis of tissue microarray further confirmed that SERP1 expression was significantly up-regulated in PDAC tissues.2.mRNA expression data of SERP1 and clinical data were obtained from TCGA database.According to the median of relative mRNA expression,PD AC patients were divided into high SERP1 expression group and low SERP1 expression group,and the relationship between SERP1 expression level and prognosis in PDAC patients was analyzed.With the increase of SERP1 expression level,the clinical stage of PD AC patients was higher,especially for T and N stages;K-M survival analysis found that patients with high SERP1 expression had shorter OS and DFS;Cox regression analysis found that the expression level of SERP1 was an independent prognostic factor.3.The result of gene set enrichment analysis(GSEA)confirmed that SERP1 overexpression was associated with cell apoptosis.Protein-protein interaction(PPI)and gene ontology(GO)found that SERP1 was associated with SRPRB/NF-?B signaling pathway.Correlation analysis showed that SERP1 expression level was positively correlated with NF-?B,and SRPRB expression level was negatively correlated with NF-?B.4.After transfecting si-SERP1 into PANC-1 cells,the expression level of SERP1 was down-regulated,and the apoptosis rate of PANC-1 cells was significantly increased.Western blot experiments confirmed that down-regulated SERP1 expression could increase SRPRB expression and SRPRB overexpression inhibited NF-?B activation,which ultimately leading to PANC-1 cells apoptosis.The results of the first part showed that the activation of STAT3,could up-regulate the relative miR-21 level in PSCs,then exosomal miR-21 was released,which was uptake by extracellular adjacent PDAC cells.Next,exosomal miR-21 activated the Ras/Erk and Ras/Akt signaling pathway by down-regulating RASA1 expression,which leading to cell migration.The results of the second part showed that PDAC patients with high SERP1 level had higher clinical stage and shorter survival time.Down-regulated SERP1 could increase the expression of SRPRB and then inhibit the activation of NF-?B signaling pathway,which ultimately promoting PANC-1 cells apoptosis.Therefore,SERP1 is expected to become a new potential prognostic marker in PDAC patients,targeting SERP1 could promote cell apoptosis.