Exploration On Reversal Of Malignant Phenotype Of Pancreatic Cancer By Wild-type Kras Gene And Lnc-TNFAIP3 And Its Mechanism

Part1 Exploration on the mechanism of wild-type KRAS in the migration and invasion of pancreatic cancerObjective:1.The purpose of this study isto investigate the effect of wild-type Kras gene overexpression on biological behavior of pancreatic cancer;2.To explore the potential molecuLar mechanism of wild-type Kras on the invasion and migration of pancreatic cancer.Methods:1.The wild-type Kras and mutant Kras overexpression genes were transfected into Panc-1 by lentivirus transfection method respectively,to construct wild-type and mutant Kras stable overexpression cell lines labeled as KrasWT and KrasG12D;2.the proliferation,migration,invasion ability ofpancreatic cancer were detected by CCK-8,colony formation,cell cycle,cell scratch assay,transwell assay,flow cytometry and so on;3.Tumor-forming ability of pancreatic cancer with different Kras gene backgrounds was detected in nude mice;4.the expression of E-cadherin,α-E-catenin,MMP-9,p-STAT3 and MMP-3 were detected by Western blot and immunohistochemical test.ResuLts:1.KranWT and KrasG12D overexpression pancreatic cancer cell lines were constructed;2.In vitro,compared with Panc-1,KrasWT was weakened in proliferation,invasion and migration,p<0.05,the difference was statistically significant,while KrasG12D invasion and migration ability was enhanced,p<0.05,the difference was statistically significant,there was no significant difference in proliferation;3.in vivo,the average tumor volume of KraaWT was smaller than that of Panc-1,and the average tumor volume of KrasG12D was larger than that of Panc-1,p<0.05,the both difference was statistically significant;4.Western blot and immunohistochemical resuLts showed that the expressions of E-cadherin,α-E-catenin,MMP-9,p-STAT3,and MMP-3 of KrasWT were up-reguLated.Conclusion:1.The wild-type Kras gene can reduce the proliferation,migration and invasion of pancreatic cancer,while the mutant Kras gene can enhance the invasion and migration of pancreatic cancer,and has a tumor-promoting effect in vivo,which is not obvious in vitro;2.The inhibition ability of wild-type Kras gene on cell migration invasion and migration may be realized throgh Wnt/β-catenin pathway.Part2 Exploration on the mechanism of lnc-TNFAIP3 in migration and invasion of pancreatic cancerObjective:1.To investigate the effect of Lnc-TNFAIP3 overexpression on pancreatic cancer;2.To investigate the potential molecuLar mechanism on the invasion and migration of pancreatic cancer.Methods:1.Lnc-TNFAIP3 stably overexpressed pancreatic cancer cell was constructed by lentivirus transfection method;2.the proliferation,migration,invasion ability were detected by CCK-8,colony formation,cell cycle,cell scratch assay,transwell assay,flow cytometry and so on;the expression of E-cadherin,α-E-catenin,vimentin and fibronectin were detected by Western blot;3.The differentially expressed genes were obtained by transcriptome sequencing and enrichment pathway analysis was performed,Genes and pathways closely related to Lnc-TNFAIP3 were selected for verification to explore the mechanism of their co-action on pancreatic cancer.ResuLts:1.Lnc-TNFAIP3 overexpression pancreatic cancer cell lines were constructed;2.Overexpression of Lnc-TNFAIP3 reduced the proliferation,migration and invasion ability of pancreatic cancer cell,and the differences in cell cycle and apoptosis were not statistically significant;3.There was no significant difference in the expression of E-cadherin,α-E-catenin,vimentin,fibronectin proteins;4.After transcriptome sequencing,2363 differentially expressed genes were obtained,1254 up-reguLated and 1109 down-reguLated.KEGG pathway analysis up-reguLated genes are mainly involved in cell cycle,RNA transport,pathogenic E.coli infection,reguLation of actin cytoskeleton,white blood cells Trans-internal migration,p53 signaling pathway,glycolysis and so on,down-reguLated expressed genes are mainly involved in amino acid biosynthesis,tumor necrosis factor signaling pathway,IL-17 signaling pathway,small analysis ribonucleic acid,insuLin resistance,complement system and other pathways.Conclusion:1.Overexpression of Lnc-TNFAIP3 can reduce the proliferation of pancreatic cancer;2.Overexpression of Lnc-TNFAIP3 decreased the migration and invasion ability of pancreatic cancer,but the expression of related proteins did not change significantly;3.Through enrichment analysis of KEGG pathway,10 genes with high correlation were screened out by cytoscape:Actb,Vcl,Fnl,Rhoa,Cfl1,Actn4,Cdc42,Wasl,Msn,Actg1 for further research.