Study On The Relationships Between Glycosylation And Malignant Behavior Of Luekemia

Objective: At present, in the study of molecular mechanism of leukemia, disease related proteins posttranslational processing mainly focused on phosphorylation and acetylated modification, the main way for protein posttranslational processing: glycosylation is seldom involved. This study would like to explore the correlation between leukemia malignant behavior and of glycosylation. Methods:(1) 183 cases of acute leukemia data is from TCGA database, big data processing method is used in studying the relevency between leukemia cell and Polypepide: N- Acetylgalactosaminyltransferases(pp- Gal NAc- Ts, EC. 2.1.41) family especially pp- Gal NAc- T1, T2 and b3-N-Acetylglucosaminyltransferases family especially β3Gn T8. Using Affymetrix Gene Chip Command Console software(version4.0, Affymetrix) processing to extract the raw data. Using Genespring software(version 12.5; Agilent Technologies) for RMA standardization and subsequent processing. Screening differential gene by T test, p value and the multiple change value. The standard of screening is raised or lowered multiples change value > = 2.0 and p < = 0.05. Gene Ontology and KEGG enrichment analysis is used to find the biological function of genes pathway mainly affected by differential genes. Using unsupervised clustering hierarchy for differential genes, research the expression patterns of differential gene between different samples.(2) Real-time quantitative PCR, Western Blot, Lectin Blot and flow cytometry are used to detection of lthe m RNA and protein expression of glycosyl transferases related to glycosylation in leukemia K562 and HL60 cells, and detect the structure N-glycan and O-glycan on cell surface. Research the correlation between these difference and leukemia clinica classification and leukemia cells malignant phenotype.(3)Collect bone marrow smears of 130 cases leukemia patient. Lectin immunohistochemical is used to detect the expression of Lactose amine polymer between different leukemia classification, different age and different gender. Lactose amine polymer is the catalysate of β3Gn T8(Gal T7) and β3Gn T2.Results:(1) The boxplot chart and the matrix chart of 183 cases of acute leukemia data shows that the homogeneity of sample data is quite well, the tendency of overall distribution data between different groups is centralized distribution, the sample of the same group concentrated in the PCA analysis chart. Using volcanic chart, screened differential genes in complex chromosome abnormalities group and gene mutation group. By Gene Ontology and KEGG enrichment analysis, find the differential genes related to glycosylation. Compared with non complex chromosome abnormalities group, the expression of β3Gn T8 is decreased in the 15;17 complex chromosome abnormalities group, involving cell components include: the cell membrane and integrated membrane, the golgi apparatus, and the expression of gene MMP2, MMP14, c- jun is increased. These genes participate jointly in MAPK pathway. Compared with non gene mutation group, the expression ofβ3Gal T1 is decreased in the pml-rar gene mutation group, involving cell components is the golgi apparatus, and the expression of geneβ3Gal T2, β3Gn T, Gn T- V is also decreased, the expression of gene pp Gal NAc-T2, ST3Gal4, ST3Gal6 is increased.(2) Real-time PCR results is the expression of β3Gn T8 and GNTⅤ in K562 cells is higher than in HL60 cells. The expression of β3Gn T8 is lower than other two genes in both two leukemia cells. The expression of β3Gn T8 in HL60 is lower than in K562. Real-time PCR results of β3Gn T8 in K562 and HL60 cells. The expression of pp Gal NAc T1 and pp Gal NAc T2 in K562 is higher than in HL60. The expression of ST6 Gal T1 in K562 is higher than in HL60. The expression of ST6 Gal T4 in K562 and HL60 is almost the same. The expression of ST3 Gal T1 inHL60 higher than in K562. The Western Blot shows that The expression ofβ3Gn T8 and pp Gal NAc T2 in K562 is higher than in HL60. The Lectin Blot shows that the expression of Lactose amine polymer in K562 is higher than in HL60. Flow cytometry results showed lectin SNA specificity identification of alpha 2, 6 connection sialic acid on K562 cells surface is lower than HL60. Lectin MAL Ⅱ specificity identification of alpha 2, 3 connection sialic acid, the expression of two cells are almost the same.(3) Detect the the expression of Lactose amine polymer of 130 cases leukemia patient bone marrow smears, it is found that in chronic leukemia, the lactose amine polymer mainly has high expression, and in acute leukemia, the lactose amine polymer mainly has low expression. Proved in clinical acute or chronic leukemia bone marrow samples, β3Gn Ts family and its catalytic products have increased expression, especially in chronic leukemia.