A Key Biomarker Of Pancreatic Cancer MiRNA And MRNA Regulatory Network,DTNA(Dystrobrevin,alpha),Affects The Biological Behavior And Mechanism Of Pancreatic Cancer

Part Ⅰ:Bioinformatics-based screening of key prognostic and diagnostic biomarkers in the miRNA and mRNA regulatory network of pancreatic cancerObjective:To screen key biomarkers in prognostic miRNA and mRNA regulatory networks based on relevant online databases of pancreatic cancer to find new potential diagnostic and therapeutic targets for pancreatic cancer.Methods:GEO2R tool was used to analyze mRNA and miRNA datasets in GEO database to obtain differential mRNA and miRNA;Kaplan-Meier Plotte was used to find prognosis-related DEpMS;miRDB,mirtarbase,targetscan7.0,encorin database were used to perform target gene The canadinate gene was obtained by GO and kegg analysis using metascape database and PPI interaction network;the difference in expression and prognostic value of canadinate gene between pancreatic cancer and normal pancreatic tissue was analyzed by GEPIA to obtain Hub gene;the Hub gene was verified by TCGA and GTEX database.The diagnostic and prognostic value of Hub gene in pancreatic cancer was verified using TCGA and GTEX databases.Results:3 miRNA and 4 mRNA datasets were downloaded from the GEO database,and the differential analysis yielded 1150 DEG.up,341 DEG.down,6 DEM.down and 24 DEM.up.9 miRNAs were associated with pancreatic cancer prognosis of which 8 were expressed up-regulated and 1 down-regulated,DEMpS predicted target genes and DEGS were taken.A total of 79 common DEMipS gene were obtained by intersection,50 were up-regulated and 29 were down-regulated.using Metascape to perform GO and kegg enrichment analysis,the common DEMipS gene was mainly enriched in:the development of the interstitial matrix,the process of enzyme-linked receptor signaling pathway,the anabolic amino acid,the signaling pathway regulating the pluripotency of stem cells and other influences.The MCODE was enriched for GO and kegg in pancreatic cancer,mainly enriched for:amino acid anabolism and regulation of stem cell pluripotency associated with cell growth and development,and 17 candidate key genes were obtained.3 key genes with significant significance in expression and prognosis were obtained from GEPIA database screening NT5E,DTNA,SERPINE1.DTNA,SERPINE1;the TCGA database verified that NT5E,DTNA,SERPINE1 had good diagnostic and prognostic value,in which SERPINE1 and DTNA were independent prognostic factors for pancreatic cancer,and mir-30b-5p/NT5E,mir-30b-5p/SERPINE1,mir-27a-3p/DTNA might affect the development of pancreatic cancer.Conclusion:In the prognostic miRNA and mRNA regulatory network of pancreatic cancer,three key genes NT5E,DTNA,SERPINE1 have good diagnostic value and prognostic significance,DTNA,SERPINE1 are independent prognostic factors in pancreatic cancer,mir-30b-5p/NT5E,mir-30b-5p/SERPINE1,mir-27a-3p/DTNA,mir-30b-5p,mir-27a-3p,NT5E,DTNA,SERPINE1 may be potential diagnostic and therapeutic targets for pancreatic cancer.Part Ⅱ:DTNA(anti-myostatin alpha)affects epithelial mesenchymal transformation,proliferation,migration,and invasive ability of pancreatic cancer through AKT signaling pathwayObjective:To investigate the effects of DTNA,a key biomarker,on the EMT,proliferation,migration,and invasive functions of pancrea tic cancer,and to investigate whether DTNA can affect the biological behavior of pancreatic cancer through the AKT signaling pathway.Methods:40 pancreatic cancer specimens were collected from the Department of Hepatobiliary Surgery of Chuanbei Medical College,an d the RNA expression levels of DTNA were detected by RT-qPCR in 40 pancreatic cancers and paired paracancerous tissues,normal and can cerous cell lines of pancreas,and the protein expression levels of DT NA were detected by WB in 40 pancreatic cancers and paracancerous tissues,normal and cancerous cell lines of pancreas.The expression le vels of DTNA protein in 40 pancreatic cancer and paraneoplastic tissue s,pancreatic normal and cancer cell lines were detected by WB;the c hanges of proliferation,migration and invasion ability of pancreatic can cer cell lines after knockdown or overexpression of DTNA were detect ed by CCK8 proliferation assay,transwell invasion and migration;the expression of EMT-related pathway proteins Ecadherin,Ncadherin and SNAIL were detected by WB after knockdown or overexpression of D TNA.The expression of EMT-related pathway proteins Ecadherin,Nca dherin,SNAIL,akt and phosphorylated protein pakt 473 in akt signali ng pathway were detected by WB.Results:The mRNA and protein expression levels of DTNA were lower in pancreatic cancer tissues and cancer cell lines than in pancre atic normal tissues and pancreatic normal cell lines.The proliferation,migration,and invasion ability of cells were diminished after overexpre ssion of DTNA in SW1990,while the proliferation,migration,and inv asion ability of cells were enhanced after knockdown of DTNA in pan c1.The data using TCGA were based on DTNA expression lines GSE A was significantly enriched in EMT,notch 1,MTOR,kras,myc and o ther signaling pathways;phosphorylation of AKT was downregulated,E MT-related pathway proteins snail and N-cadherin were downregulated and E-cadherin was upregulated after overexpression of DTNA in SW1990,and vice versa.PANC1 knockdown of DTNA resulted in upregula tion of AKT phosphorylation,downregulation of EMT-related pathway proteins snail and N-cadherin,and upregulation of E-cadherin.Conclusion:The expression of the key biomarker DTNA was dow n-regulated in cancer tissues and pancreatic cancer cells,and for the fi rst time,DTNA was identified as an oncogene in pancreatic cancer tha t can affect the EMT,migration,invasion,and proliferation ability of pancreatic cancer cell lines by regulating the activation of AKT signali ng pathway.Part Ⅲ:Validation of the relationship between miR-27a-3p expression and prognosis in pancreatic cancer and miR-27a-3p targeted regulation of DTNAObjective:To verify the clinical significance of miR-27a-3p in pa ncreatic cancer and whether miR-27a-3p can target and regulate DTNAMethods:40 tissue specimens of pancreatic cancer patients from t he Department of Hepatobiliary Surgery of Chuanbei Medical College were collected.RT-qPCR was performed to detect the RNA expression levels of miR-27a-3p in 40 pancreatic cancer cases and paired paraca ncerous tissues,normal and cancerous cell lines of the pancreas.expre ssion correlation;using encorin database to validate the prognostic sign ificance of mir-27a-3p and pancreatic cancer;Targetscan predicted 2 re gulatory sites of miR-27a-3p targeting DTNA in PANC1 cell line using dual luciferase reporter gene assay to validate the targeting and regula tory relationship between miR-27a-3p and DTNA;in After overexpressi on of miR-27a-3p in PANC1 cell line or inhibition of miR-27a-3p exp ression in sw1990 cell line,the expression level of DTNA was detecte d by RT-qPCR with WB.Results:mir-27a-3p expression was elevated in pancreatic cancer t issues compared to normal pancreatic tissues;expression of mir-27a-3p and DTNA expression were negatively correlated in both encorin and pancreatic cancer tissue specimens in the online database;DTNA expr ession was elevated after knockdown of mir-27a-3p in SW1990 by RTqPCR and WB,and overexpression of mir-27a-3p in PANC1.DTNA e xpression was decreased after overexpression of mir-27a-3p in PANC1;two binding sites between mir-27a-3p and DTNA were predicted by T argetscan,and dual luciferase assays confirmed that mir-27a-3p directly targeted the WT2 sequence site in the UTR3’ end of DTNA.Conclusion:mir-27a-3p was highly expressed in pancreatic cancer tissues and pancreatic cancer cell lines,and mir-27a-3p could target a nd regulate DTNA.