| Pancreatic cancer and Cholangiocarcinoma are common malignant tumor of digestive system, due to the low diagnosis rate of early tumor, most of patients has been found in advanced stage of tumor. There is lack of effective treatment, survival rate is low. So improving early diagnosis rate and the intervention of potential targets are particularly important to pancreatic cancer and Cholangiocarcinoma.N- glycosylation is an important way of post-translational modification of proteins, resulting in changes of glycoprotein function of cell surface receptors and adhesion molecules such as E- cadherin and integrin, these changes are closely related to cancer cell phenotype. Glycosylation is catalysed by different glycosyl transferase, the activity of glycosyltransferase and gene expression of enzyme change in diseases such as cancer. Therefore, the analysis of N- glycan structure and the analysis of the corresponding glycosyl transferase are expected to find biomarkers for early diagnosis of pancreatic cancer and cholangiocarcinoma, and find the role of glycosyltransferases in the development of pancreatic cancer and cholangiocarcinoma. This study establishs early diagnosis model on N- glycan and the existing clinical tumor marker. The corresponding glycosyltransferase expression in tissue of malignant biliary and pancreatic tumors were studied, and the further study about the effect of glycosyltransferases on cell lines in vitro and in vivo was done to look for potential intervention targets.Part 1: N-glycomic study in pancreatic cancer and cholangicarcinoma patientsObjective: Identify and verify biomarkers based on N- glycan analysis, combini ng with the existing clinical tumor markers to improve the early diagnosis rate.Methods: Use DSA-FACE to analysis N- glycan of serum of 164 cases(including 72 cases of pancreatic cancer, 26 cases of pancreatitis and 66 cases of sex, age matched healthy controls), serum of 171 cases(including 59 cases of cholangiocarcinoma, 32 cases of bile duct calculus, 20 cases of bile duct cyst and 60 cases of healthy control). Compare the differences between groups, establish diagnosis models on logistic regression method,and evaluate the effect of diagnosis models using ROC curve. Results:1. pancreatic cancer:(1) Compared with the normal control group, four N-glycan structure(NGA2F, Peak1; NA3 Fb, Peak 9; Peak 10; NA3 Fc, NA4 Fb, Peak 12) were increased, three N-glycan structure(NG1A2F, Peak NA2 F, 4; Peak 6; NA3 8, Peak) were down in the patients with pancreatic cancer.(2) Establish a diagnosis model Glyco PCtest =(peak 9+10+12/peak 4). In the Training Cohort area under ROC curve of Glyco PCtest was 0.859, when the optimal cut-off value was 0.97, the corresponding sensitivity and specificity were respectively 69.44%, 89.39%. In the validation group sensitivity and specificity of Glyco PCtest were 75%, 82.76%.(3) Combining with tumor marker CA19-9, establish a diagnosis model PCmodle=2.376 Glyco PCtest+0.052CA19-9-4.075, in the Training Cohort area under ROC curve was 0.937, when the optimal cut-off value was-0.06, the corresponding sensitivity and specificity were respectively 80.56%, 98.48%. In the validation group sensitivity and specificity were 91.67%, 89.66%.2. cholangiocarcinoma:(1) Compared with the normal control group, three N-glycan structure( Peak 9; NA3 Fc Peak 10; NA4 Fb, Peak 12) were increased, two N-glycan structure(NA2F, Peak 6; NA2 FB, Peak 7) were decreased in patients with cholangiocarcinoma.(2) Establish a diagnosis model Glycomodel1=10.183Peak10+ 3.232Peak12+0.073CA19-9-8.381 to identify cholangiocarcinoma and normal control. The area under ROC curve of Glycomodel1 was 0.977, when the optimal cut-off value was 0.552, the corresponding sensitivity and specificity were respectively 91.5%, 98.3%.(3) To distinguish the calculus of bile duct and cholangiocarcinoma, establish a diagnosis model Glycomodel2=9.442Peak10+0.009CA19-9-4.390, The area under ROC curve of Glycomodel2 was 0.925. When the optimal cut-off value was 0.648, corresponding sensitivity and specificity were respectively 81.4% and 93.8%.(4) To distinguish the choledochal cyst and cholangiocarcinoma, The area under ROC curve of Peak10 was 0.923. When the optimal cut-off value was 0.485, the corresponding sensitivity, specificity were respectively 71.2%, 100%.Conclusion: The diagnosis model basing on N- glycan can be used as a marker for early diagnosis of pancreatic cancer and cholangiocarcinoma. combining with tumor markers CA19-9 can improve the diagnosis efficiency of N- glycan.Part 2: Role and related mechanism of the N-acetylglucosaminyltransferase-V in biliary and pancreatic cancerObjective: N-Acetylglucosaminyltransferase-V(Gn T-V) is a glycosyltransferase encoded by the Mgat5 gene that catalyzes the formation of β1,6Glc NAc(N-acetylglucosamine) branches on N-glycans. It is believed to be associated with cancer growth and metastasis. The role of GNT-V in pancreatic cancer and choangiocarcinoma is unclear. In this study we investigated the effects of GNT-V on tumor growth and metastasis both in vivo and in vitro.Methods: Reverse transcriptase-polymerase chain reaction(RT-PCR) was conducted to evaluate the expression of GNT-V using 19 pairs of tumor and adjacent tissues. RT-PCR expressions were confirmed by Lectin blot. The Lenti-sh RNA vector system was constructed, packed, and purified. Then, cell growth, wound-healing, cell invasion assays were conducted to examine the effect of GNT-V knockdown in ASPC-1, PANC-1, Bx PC-3, CFPAC-1 cells and HCCC-9810. Flow cytometry analysis was performed to observe the effect of GNT-V knockdown on cell cycle. Xenograft model were established to examine the effect of GNT-V knockdown on tumor formation in vivo. Western blot was adopted to observe the effect of GNT-V knockdown on Mitogen-activated protein kinase(MAPK) signaling.Results: 1. Pancreatic cancer:(1)The results showed that the expression of GNT-V was lower in PC tissues compared to adjacent tissues. Total Glc NAc abundance also trended to be lower in pancreatic tumor compared to that in paired adjacent tissues.(2) Cell growth, wound-healing, cell invasion assays showed that knockdown of GNT-V promoted PC cells proliferation, migration, and invasion. Furthermore, knockdown of GNT-V promoted S-phase and G2-phase of cell cycle. GNT-V knockdown accelerated tumor formation and increased tumor weight in nude mices.(3) Given the evident effects of GNT-V on PC cells growth and metastasis, signaling pathways involved in tumor growth and metastasis that might be activated by GNT-V were analyzed. The level of phosphorylation of p38 Mitogen-activated protein kinase(P-p38MAPK) was increased by GNT-V suppression.2. cholangicarcinoma:(1) Cell growth, wound-healing, cell invasion assays showed that knockdown of GNT-V inhibited HCCC-9810 cell proliferation, migration, and invasion.(2) Signaling pathways involved in tumor growth and metastasis that might be activated by GNT-V were analyzed. The level of phosphorylation of SAPK/JNK was increased by GNT-V suppression.Conclusion:(1) Knockdown of GNT-V improved PC carcinogenesis and metastasis by activating p38 MAPK signaling pathway.(2) Knockdown of GNT-V inhibited carcinogenesis and metastasis by activating SAPK/JNK signaling pathway in cholangicarcinoma. |
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