Screening And Functional Study Of Trypsinogen Gene Mutation In Peripheral Blood Of Patients With Pancreatic Cancer

Objective: The pathogenesis of pancreatic cancer is very complicated and the effect of diagnosis and treatment is extremely poor.Unfortunately,there is no specific mechanism for the tumorigenesis,proliferation and metastasis of pancreatic cancer(PC).Protease Serine 1(PRSS1)mutations can trigger chronic pancreatitis,and PRSS1 mutations have been also identified in peripheral blood of patients with pancreatic cancer in previous studies.But it has not yet been explained whether the PRSS1 mutation is a primary factor,and whether the PRSS1 mutation promotes the malignant proliferation and metastasis of pancreatic cancer.Therefore,in this study,the frequency of PRSS1 mutation in peripheral blood of pancreatic cancer patients was detected by direct sequencing,and the human mutant gene(PRSS1_p.Thr137Met)was knocked in the mouse model(mt PRSS1)to verify the relationship between mutant trypsin and pancreatic cancer.And explore its possible pathogenesis.Methods:(1)Extraction of blood DNA from 80 cases PC patients and 220 cases control group,the mutation and polymorphism site were located and the mutation types were analyzed by direct sequencing,after PCR amplification.(2)Analysis the tissues of pancreatic cancer of PRSS1 mutation by immunohistochemical.(3)Human mutant gene knock in mice(mt PRSS1)were obtained by transgenic technique.Then reproduced and identified the genotypes of offspring.(4)Analysis the differences between mt PRSS1 and wild type(WT)by biochemical indicator,H&E,immunohistochemical,immunofluorescence and specific stain of pathology.(5)Select 3 mt PRSS1 mice and 3 WT 8-week old littermates,then carried outexperiments of extraction of pancreas tissue RNA for q RT-PCR and western blot to verify possible protein pathways.Results:(1)We found 4 cases with PRSS1_p.Thr137 Met mutation,1 cases with PRSS1_p.Gln56* mutation(which is newly discovered mutation and stops codes appear early and produce truncated proteins),1 case with PRSS1_p.Asp162 Asp in all the patients with PC,while no mutations were found in the corresponding region of the PRSS1 gene in the control group.(2)The expression of trypsin in pancreatic cancer tissues is higher than paracancerous tissue.(3)Human mutant PRSS1 gene knockin mice(mt PRSS1)were successfully constructed,and the offspring mice(F1-F6)grew well.The two genetypes(mt PRSS1 and wild genotypes)were identified and accorded with Mendelian law in the offspring mice.(4)The levels of lipase(mt PRSS1:441±69.376 U/L;WT:260±8 U/L;P=0.0109),serum amylase(mt PRSS1:1953.333±298.719 U/L;WT:1073.333±16.073 U/L;P=0.0073)and blood glucose(mt PRSS1:15.433±1.464 mmol/L;WT:4.933±0.757 mmol/L;P=0.0004)in mt PRSS1 mice were significantly higher than those in normal mice.(5)Histopathology showed that the small pancreatic ducts in mt PRSS1 mice were abnormal in structure and cells,the epithelial cells of ducts were disordered,mucous metaplasia and pancreatic ductal intraepithelial neoplasia(Pan IN).(6)The results of Western blot showed that the level of DDIT4、PI3K、m TOR、MMP(MMP1,MMP8 and MMP9)in mt PRSS1 mice were higher than those in WT mice,p53 was lower than it in WT mice.(7)The q RT-PCR showed that DDIT4,STAT(STAT5A and STAT6),JAK(JAK1 and JAK2)in pancreatic tissues of mt PRSS1 mice were all increased and concentrated on the JAK-STAT path.Conclusion:(1)There were PRSS1 gene mutations in the peripheral blood of patients with pancreatic cancer,of which PRSS1_p.Thr137 Met was a relatively high frequency mutation(5%).(2)The early changes of pancreatic cancer in mt PRSS1 mice were similar to those in human pancreatic cancer.PRSS1_p.Thr137 Met may lead to pancreatic damage,inflammation,and changes in indicators of tumor.(3)PRSS1_p.Thr137 Met leads to increased expression of the DDIT4 gene,and may activate both PI3K-Akt and JAK-STAT signaling pathways to cause pancreatic cancer at the same time.Invasion and metastasis occur through MMPs.