| Objective:The incidence of pancreatic cancer in our country is about 6.22/100,000,which is one of the top ten malignant tumors.Its survival rate is almost the worst among 60 common tumors.Unfortunately,the specific mechanism of pancreatic cancer have not been clarified.Pancreatic cancer patients have mutations in trypsinogen gene.As a signal molecule,trypsin can activate PAR2 to stimulate the proliferation and adhesion of pancreatic cancer cells.It can also participate in the invasion and metastasis of pancreatic cancer by degrading the extracellular matrix.The purpose of this study is to screen Protease Serine 1(PRSS1)mutations in peripheral blood samples selected from pancreatic cancer patients,then to disclose the impact of common mutation form of PRSS1(PRSS1_R116C)on the occurrence and development of pancreatic cancer through cells and transgenic mouse models.Methods:(1)Two families of pancreatic cancers were screened with full exon sequencing.The subjects in the family were matched as: two pancreatic ductal adenocarcinoma patients and three healthy first-degree relatives in one family,one pancreatic ductal adenocarcinoma patient,one chronic pancreatitis patient,and two healthy first-degree relatives in another family.(2)Screening out pathogenic genes by combining genetic model and analysising sequencing results.(3)Verifying the possible pathogenic genes by designning primers and sangar sequencing in clinical patients.(4)Pancreatic Cancer Cell Line(PANC-1)with PRSS1_R116C mutation was constructed.(5)RNA-SEQ high-throughput sequencing was done to detect the effect of PRSS1_R116C on Pancreatic cancer cells.(6)Through KEGG enrichment analysis of differential genes screened by RNA-SEQ.(7)Major differential proteins or genes were verified by phosphorylated antibody chips and WB.(8)Knock-In mouse model was constructed: i ZEG-mt Prss1 and i ZEG-wt Prss1 randomly inserted into the mouse genome to obtain over expressed transgenic mice by using the system of Piggy BAC transposase and the way of injecting fertilized egg.(9)Observing pancreatic tissues of transgenic mice and detecting tumor related proteins.Results:(1)Full exon screening: In the total single nucleotide polymorphism(SNP),there are 14,246 synonymous mutations and 13,612 missense mutations in the coding region.There are 39 SNPs which changed the termination password into non-stop password,132 SNPs changed the password into a stop password,24 SNPs changed the starting password into a non-start password and 91 SNPs changed the splice acceptor or splice donor in the splice site region.In the overall In Del,there are 228 frameshift mutations in the coding region,4 In Dels changed the termination password into a non-terminate password,2 In Dels changed the start password changed into a non-starting password,and 43 In Del changed in the splice site area.We filtered the mutations in the normal individuals,and then screened for gene mutations which associated with cancer or Immunity.Finally,we obtained 586 gene mutations including PRSS1.Previously,We have reported that PRSS1 gene mutation have been found in peripheral blood of patients with pancreatic cancer.It is important that PRSS1 mutation have genetic tendency and explicit rate.Therefore,we focus our verification on PRSS1.(2)7 different mutations of the PRSS1 gene(all are heterozygous mutation)were found in the peripheral blood of 86 patients with pancreatic cancer:(1)8 patients carried p.Thr137 Met mutation(which may influence the combination of calcium).(2)2 patients carried p.139 Cys>Cys synonymous mutation.(3)4 patients carried p.R116 C mutation(which may influence fold of trypsinogen)(including 2 patients from full exon sequencing).(4)2 patients carried V123 M mutation(function unknown).(5)5 patients carried IVS 3 +110 C>T(function unknown).(6)1 patients carried p.13 Leu>Leu mutation(function unknown).(7)1 patients carried PRSS1_Q56*,a new mutations form(function unknown).(3)Analysis of differential gene between Pancreatic Cancer Cell Lines which overexpressed the mutated PRSS1 gene(PRSS1_p.R116C)and the Wild PRSS1 by RNA-SEQ.We found that the expression of interleukins(IL2,6,7,8 and 13),collagen(COL4 and COL9)and tumor-associated proteins(k Ras,MAPK,PI3 K,TGF,RB1)were all up-regulated,while the expression of JUN,MMP1,MMP9 and MMP14 were down-regulated.(4)In the result of WB,it was found that DNA damage-inducing transcript 4(DDIT4)and m TOR were highly expressed in PRSS1_R116C pancreatic cancer cells compared with pancreatic cancer cells which overexpressed wild-type trypsin.(5)The proportion of S phase in PANC-1 cells(PRSS1_R116C)was(34.69±1.69)%,while which in PANC-1 cells overexpressed wild type PRSS1 was(36.63±1.97)%.The difference between the two is not obvious,but both were higher than normal PANC-1 cells(24.57±5.40)% which not overexpressed trypsin.At the same time,there was no significant difference in G2/G1 ratio between groups,suggesting that high trypsin can arrest PANC-1 cells in S phase without affecting G1/G2 conversion;and PRSS1_R116C mutation itself does not affect cycle,just could cause the overexpression of trypsin and drive the cell cycle to change after mutation.(6)In the transgenic mouse model,the pancreas of PRSS1_R116C mice was significantly enlarged and the texture was hard.Histopathology showed that pancreatic ductitis was present in PRSS1_R116C transgenic mice(EIIA and PDX1).Inflammation in the pancreatic ducts was followed with structural and cellular atypia,disorganization of ductal epithelial cells,mucinous,and intraepithelial neoplasia(Pan INs).Islet aggregation was found in many areas,which looks like as tissue hyperplasia.Conclusions:(1)PRSS1 mutation is one of the important marker genes of pancreatic cancer.The total frequency of mutation is about 24.4%.Most of the mutations occur in exon 3.(2)PRSS1_R116C mutation can promote the proliferation and metastasis of pancreatic cells.(3)PRSS1_R116C mutation can cause pancreatic ductal epithelial dysplasia or intraepithelial neoplasia(Pan INs). |
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