Aberrant Glycosylation Of N-Acetyllactosamine And Their Roles In Progression Of Gastric Cancer

Background:Gastric carcinoma(GC)is common malignant tumor,that endangers human health.Glycosylation is an important posttranslational modification.It well known that the glycosylation of protein is altered during the process of tumor development and metastasis.However,the roles of GC associated aberrant glycosylation which play in tumor process need to be clarified.Hence,the aberrant glycosylation which associated with GC and the synthesis pathway of aberrant glycosylation will be characterized in this study.Subsequently the effects of abnormal glycosylation on the invasion and metastasis of GC cells were analyzed.This study will provide help to understand the fuction of abnormal glycosylation in tumor development.Methods:1.The glycan profiles of glycoproteins from GC cells and tissue are characterized by lectin microarray,and the abnormal glycopatterns associated GC are selected and confirmed by lectin histochemistry and lectin cytochemistry on cellular level;2.The(Maackia amurensis I,MAL-I)-Magnetic particle complex is constructed and used to isolate glycoproteins from GC cells/ normal cells as well as GC tissue/ normal tissue.The N-glycans are characterized compared by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS);3.The transcriptional level of glycan related genes from tumor tissues with different TNM stages and their corresponding controls are investigated by glycan related gene microarray.After comparison,the glycan related genes which show up-regulated or downregulated are selected.The expression level of the altered gene is analyzed by using the data from TCGA and GEO database;4.Construction of shRNA and over expression plasmid vectors,and establishment of stable transfected GC cell lines.Assessment the effect of B4GALT3 on the motility and invasion ability of GC cells by using wound healing assay and transwell assay.Results:1.The glycan profiles of glycoproteins from GC cell lines and GES-1 were compared by lectin microarray,the results indicated that the relative abundance of glycopatterns recognized by MAL-I、ACA and SNA showed significantly increased in all GC cell lines compared to GES-1.The results of lectin cytochemsitry also indicated that glycopatterns recognized by MAL-I showed Abnormal accumulation in cytomembrane and ECM in GC cells,which was consisted with the results of lectin microarray.2.The glycan profiles of tissue proteins from tumor and normal cases were also assessed by lectin microarray.The results showed that the relative abundance of glycopattern recognized by MAL-I exhibited a coincidentally increased trend during the tumor progression.Furthermore,the relative of abundance of this glycopattern showed increased in tumor samples at stage III compared early stage(stage I and stage II),and exhibited increased in lymph node positive tumor cases compared to lymph node negative tumor cases,which indicated the abnormal expression of this glycopatterns correlated with advance stage and lymphatic metastasis.The results of lectin histochemsitry showed that the staining intensity of MAL-I showed increased in tumor cases and enriched in region of cytomembrane and ECM.These results were consisted with results of lectin microarray.3.The results of MALDI-TOF-MS indicated that LacNAc-contained N-glycans exhibited a growing trend during the tumor development.It’s worth noting that,LacNAc contained N-glycans which characterized in tumor samples in the early stages(stage I and II)did not show significant change.However,more LacNAc contained N-glycans were characterized in tumor tissue with the advanced stage(stage III)compared to early stages.Moreover,10 neo-LacNAc-contained N-glycans that were identified and annotated with proposed structures were unique in GC samples with advanced stage.Five of which were modified with sialic acid and two of which were modified with fucose based on this glycopattern to form sialylated N-glycans or lewis antigen.4.The transcriptional level of glycan related genes from tumor and normal tissues were evaluated by gene microarray.The results showed that the transcriptional level of B4GALT3 showed significantly increased in tumor samples at stage II and III compared to the corresponding controls.Interestingly,the glycosyltransferase of b4galt3 which encoded by B4GALT3 is responsible for the synthesis of LacNAc and poly LacNAc.TCGA and GEO database were used to investigate the expression of B4GALT3 in large-scale of tumor cases.As a result,the expression level of B4GALT3 showed significantly increased in GC patients compared to normal controls.Our results indicated the expression of B4GALT3 was increased in tumor tissue,moreover,it also showed increased in advance stage of GC patients compared to the patients at early stage.Therefore,it concluded that the expression level of b4galt3 was increased in GC development,which promote the synthesis of LacNAc and led to the abnormal accumulation of LacNAc in regions of cytomembrane and ECM of GC cells.5.The lentivirus plasmids were transfected into 293 T cells to generate lentivirus particles.The virus was added into cells and the stably transfected cells was selected by puromycin and flow cytometer.It showed that the expression of B4GALT3 has no effect on proliferation ability of SGC-7901,however,it could promote migration,invasion and colony formation of SGC-7901.Conclusion:Our findings revealed that the glycopatterns of LacNAc recognized by MAL-I showed abnormal accumulation in in regions of cytomembrane and ECM of GC cells.Moreover,the aberrant expression of LacNAc correlated with advance stage of GC and lymphatic metastasis.The over expression of b4galt3 contributed to the abnormal accumulation of LacNAc.Modulating B4GALT3 expression in GC cells directly altered the ability of migration and infection of GC cells.This study provides a new sight to investigate the fuctions of the aberrant of glycosylation in tumor development and metastasis,and provides theoretical basis for illumination of molecular mechanism of GC.