The Function Of ?3GnT8 And Polylactosamine In The Malignant Phenotype Of Liver Cancer

Part I The expression of ?3GnT8 and releated genes,polylactosamine in hepatocellular carcinomaObjective:To detection the expression of ?3GnT8,the related genes and polylactosamine in liver cancer cell lines and hepatocellular carcinoma.Methods:(1)qPCR method was used to detect mRNA expression in multiple cell lines,such as Lo2,QSG7701,SMMC7721,SK-HEP-1 and HepG2 cells.(2)Detect the protein expression of ?3GnT8 in SMMC7721,SK-HEP-1 and HepG2 cells by western blot assay.(3)Analysis of the expression of ?3GnT2,?3GnT8,GnTV and c-jun in human hepatocellular carcinoma andpara-carcinoma tissues by qPCR method.(4)Immunohistochemical technology was conducted to analyze ?3GnT8,GnTV,c-Jun and polylactosamine expression level in human hepatocellular carcinoma and para-carcinoma tissues.Results:(1)?3GnT8 mRNA expression level of liver cancer cell lines is remarkably higher than that in nomarl liver cell lines;the expression of(33GnT2 has no relation with ?3GnT8;the expression of c-jun,GnTV and ?3GnT8 present the same tendency in different cell lines.(2)?3GnT8 level of liver cancer tissue is higher than that of para-carcinoma tissue,and GnTV,c-Jun,polylactosamine expression level take on the consistent tendency with ?3GnT8.Conclusion:The differential expression of ?3GnT8,the related genes and polylactosamine in liver cancer cell lines suggests that ?3GnT8 may be regulated by c-jun and play an important role in the synthesis of polylactosamine in hepatocellular carcinoma.Part ? The role of ?3GnT 8 expression in themalignant phenotype and glycosylation of hepatocellular carcinomaObjective:To investigate the function of ?3GnT8 in malignant phenotype of hepatocellular carcinoma,and analysis of the structure of polylactosamine that synthesised by ?3GnT8,the level of CD 147 and the carbohydrate chain in liver cancer cells.Methods:(1)The ?3GnT8 overexpression liver cancer cell lines were established using lentivirus system,and then,transfection efficiency was detected with fluorescence microscope.?3GnT8 protein level was analysised with western blot.(2)The expression of ?3GnT8 was down-regulted in liver cancer cells by ?3GnT8 interference vector.(3)Co-IP was used to analyze the interaction of ?3GnT8 and CD 147.(4)Western blot and lectin blot were used to detect the polylactosamine and CD 147 level in the ?3GnT8 overexpression cells and cells that transfected with ?3GnT8 interference vector.(5)Transwell assay was conducted to detect the influence of migration/invasion ability in?3GnT8 overexpression and lowexpression cells.The relationship between ?3GnT8 and tumorigenic ability of cells was detected with nude mouse transplantation tumor experiment.(6)Lectin microarray was used to analyze theexpression of carbohydrate chainin ?3GnT8 overexpression cells.(7)MALDI-TOF mass spectrometry was used to analyze the N-glycanstructure of ?3GnT8 overexpression cells.Results:(1)Polylacsamine and HG-CD147 level are respectively increased and declined in ?3GnT8 overexpression and ?3GnT8 interference cells.(2)Overexpression and interference of ?3GnT8 can respectively increase and decrease migration and invasion ability of cells.?3GnT8 can increase tumorigenic ability of liver cancer cells in nude mice.(3)The protein of ?3GnT8 and CD 147 can bind together in SK-Hep-1 and HepG2 cells.(4)Overexpression of ?3GnT8 increases the polylacsamine,Sialyl glycan,Fucosylated chain and N-glycansby lectin microarray assay.(5)Overexpression of?3GnT8 increases the abundance of N-glycan and potential polylacsamine structure in liver cancer cells by MALDI-TOF.Conclusion:(33GnT8 regulates the expression of polylactosamine and glycosylation level of CD 147,and then influces the malignant phenotype of liver cancer.In conclusion,?3GnT8 plays an essential role in the invasion and migration of liver cancer by synthesis the polylactosamine structure and changing CD 147glycosylation.Part III The role of c-jun in the regulation of ?3GnT8 expressionObjective:To investigate the function of c-jun in the regulation of ?3GnT8.Methods:(1)ChIP assay was conducted to detect the interaction between c-Jun and ?3GnT8 promoter region.(2)The ?3GnT8 overexpression liver cancer cell lines were established using lentivirus system,and then,transfection efficiency was detected with fluorescence microscope.?3GnT8 protein level was analysised with western blot.(2)Establish the liver cancer cell lines overexpression of c-jun and lowexpression of c-jun.(3)mRNA,proteion,polylactosamine,potential of migration and invasion were examined after the cells transfected with c-jun overexpression and interference vector.(4)?3GnT8 overexpression cells were cotransfection with c-jun interference vector,and CD 147,polylactosamine,migration and invasion ability were analyzed.Results:(1)ChIP assay shows that transcription factor c-Jun can bind to the promoter region of ?3GnT8 gene in SK-Hep-1 and HepG2 cell lines.(2)Expression of?3GnT8,HG-CD147,and Polylactomine can be regulated by overexpression and interference of c-jun,which will improve and reduce migration and invasion ability of cells respectively.(3)Cotransfection assay of ?3GnT8 overexpression cells with c-jun interference vector proves that lower c-jun expression will reduce ?3GnT8 level,and finally,polylactomine,glycosylation level of CD 147 and migration/invasion ability was also decline.Conclusion:(1)The migration and invasion capability can be regulated by transcription factor c-Jun.(2)Transcription factor c-Jun can regulate the expression of P3GnT8.Combining the character of ?3GnT8 in liver cancer and regulation effect of migration and invasion by c-Jun in liver cancer,we can conclude that migration and invasion ability regulated by c-Jun can be realized through the pathway of regulating the expression of ?3GnT8 which synthesis the formation of polylactosamine and changing level of glycosylation of CD 147.In conclusion,transcription factor c-Jun can alter cell migration and invasion of liver cancer.And the regulation of ?3GnT8 by c-Jun may be one of the mechanisms that c-Jun regulates the migration and invasion of liver cancer.Because of the multiple regulation effects of c-Jun,the regulation effect of migration and invasion ability through ?3GnT8 may be only one of numerous pathways.Detailed regulatory mechanism should be researched farther.