Study On The Characteristics And Clinical Significance Of Abnormal Glycosylation Modification In Gallbladder Carcinoma

Part ?: N-glycomic study in gallbladder carcinoma patientsGallbladder carcinoma(GBC)is the most common cancer in biliary tract malignant tumor.For the ineffective method of early diagnosis and the high degree of malignancy,the prognosis of patients with GBC is still pretty poor.In recent years,the study of glycomics based on the abnormal glycosylation modification of protein has established a new method in the research of noninvasive tumor markers.We had found that the characteristic changes of sugar chains in serum of patients with colorectal cancer,gastric cancer,pancreatic cancer,hepatocellular carcinoma and multiple myeloma based on the DNA sequence-assisted fluorophore-assisted carbohydrate electrophoresis(DSA-FACE)technique,and we had constructed a series of N-glycans markers for the identification and diagnosis in those diseases.On account of the previous researches,this project was aimed to explore the changes of N-glycans of serum in GBC and construct a highly specific and(or)high sensitivity diagnostic model as a warning indicators for GBC.The N-glycans in serum from GBC,chronic cholecystitis(CP)and healthy control(HC)was tested by DSA-FACE technique.The analysis of serum indicated that:(1)The biantennary glycan(peak4-peak6)of serum N-glycans profiling were decreased and the tri-or tetra-antennary glycan(peak9-peak12)of serum N-glycans profiling were increased in GBC compared with CP and HC;(2)There was no correlation between the peak of N-glycans and CEA,while peak9'and peak10 were positively correlated with CA19-9;(3)In the differential diagnosis of GBC and HC,the area under curves(AUC),sensitivity,accuracy and Youden's index of the multi-parameters diagnostic models named GBCglyco A(GBCglyco A = 0.077 × peak1 + 0.983 × peak9' + 1.212 ×peak12)(0.911?80.85%?85.11%?0.70)were improved compared with CEA(0.749?26.66%?63.83%?0.27)and CA19-9(0.854?59.57%?79.79%?0.60)alonely;In the differential diagnosis of GBC and CP,the AUC,sensitivity,accuracy and Youden's index of GBCglyco B(GBCglyco B = 0.001× CA19-9 + 4.429×peak12+2.042)(0.844?74.47%?78.72%?0.57)were improved compared with CEA(0.783?26.66%?63.83%?0.27)and CA19-9(0.764?59.57%?70.21%?0.40)alonely;(4)In the differential diagnosis of GBC and HC with CA19-9 negative,the AUC,sensitivity,accuracy and Youden's index of GBCglyco A(0.865?73.68%?85.93%?0.63)were also improved compared with CEA(0.71?15.79%?75.76%?0.16);what's more,in the differential diagnosis of GBC and CP with CA19-9 negative,the AUC,sensitivity,accuracy and Youden's index of GBCglyco B(0.792?57.89%?80.70%?0.50)were also improved compared with CEA(0.754?15.79%?75.76%?0.16);(5)As for the diagnosis of lymph node metastasis of GBC patients,the AUC,sensitivity,accuracy and Youden's index of peak11(0.739?76.92%?76.60%? 0.53)were significantly higher than that of CA19-9(0.545?46.15%?70.72%? 0.26),but the specificity of peak11 was similar with CA19-9.Although the sensitivity of CEA was 100%,but the specificity(25.53%)was poor,and the Youden's index of CEA was only 0.26.Conclusion: the tri-or tetra-antennary glycan of serum in GBC were increased,and GBCglyco A and GBCglyco B may provide a more sensitivity diagnostic index for GBC patients,especially the differential diagnosis for GBC patients with CA19-9 negative.What's more,the change of the expression level of peak11 can be used as an assistant index for patients with GBC.Part ?: Biological function and related pathogenesis of N-acetyltransferase V in gallbladder carcinomaGlycosylation,the most complex and diverse structure in protein post-translational modification,is the reaction in which a carbohydrate is attached to the specific amino acid residues of specific amino acid sequences on protein by specific glycosyltransferase and glycosidase.N-Acetylglucosaminyltransferase V(Gn T-V),catalyzing the reaction of adding ?1,6-N-acetylglucosamine(Glc NAc)on asparagine-linked oligosaccharides of cell proteins,was one of the first glycosyltransferases for which the transcriptional regulation pathway was determined.It was reported that Gn T-V was crucial for the malignant degree and prognosis of tumor for instance hepatocellular carcinoma and breast cancer.In addition,we found that the tri-or tetra-antennary glycan of serum N-glycans profiling were increased apparently in GBC in the former research.So,this study was aimed to explain biological characteristics of GnT-V on GBC cells in vitro and in vivo and to reveal its potential molecular mechanism.Firstly,we investigated the expression of Gn T-V and its production using GBC tissues by lectin blot(LB)and Western blot(WB)method.Then the Gn T-V knockdown cell line by a small interfering RNA was constructed with the cells which had the highest expression level of Gn T-V.Finally,the cell function tests,nude mice bearing tumor experiment and transcriptase sequencing analysis were adopted to observe the effect of Gn T-V in GBC cells.(1)Histological study showed: by comparison with adjacent non-tumor tissues,the expression of RNA and protein of Gn T-V and its production were improved in GBC tissues.It was speculated that the increased expression of the tri-or tetra-antennary glycan of GBC serum N-glycans profiling may be related to the increased expression of Gn T-V;(2)Cell function tests revealed: reducing expression of Gn T-V inhibited the proliferation,migration,invasion abilities of GBC cells.Meantime,cell cycle progression was inhibited with the interfering cells;(3)Nude mouse model demonstrated: Down regulation of Gn T-V inhibited the growth of tumor in vitro;(4)The results of transcription sequencing Indicated: there were 23609 co-expression genes between interfering cells and mock cells.Further screening,we got the 6differentially expressed genes(DEGs),namely CGA,EGR1,HAL,PCK1,RASSF9 and TNNT2 gene.We found that CGA and RASSF9 gene were highly expressed in cancer tissues and EGR1 gene was highly expressed in the adjacentt non-tumor tissues,while the expression of HAL,PCK1 and TNNT2 gene were not significantly different in these two groups.The KEGG pathway analysis showed that the gene was mainly enriched in the signaling pathway such as Mitogen-activated protein kinase(MAPK)signaling pathway.Conclusion: The expression of GnT-V and its products were improved in GBC which may promote the proliferation,invasion,migration and tumorigenicity abilities of GBC cells by affect the expression of MAPK and other signaling pathways.