The Roles And Molecular Mechanism Of ST6Gal-I In Human Hepatocellular Carcinoma Tumorigenesis And Development

Hepatocellular Carcinoma is a common malignant tumor.Although the medical level develops unceasingly,the data about the International Agency for Research on Cancer showed that hepatocellular carcinoma(HCC)is the fifth most frequently diagnosed cancer.Nowdays,alpha-fetoprotein(AFP)is still the most common tumor marker in early diagnosis of HCC,but only 30–40% of HCC patients are diagnosed in the early stage and are therefore eligible for potentially curative treatment such as resection,transplantation,or local ablation.Demonstrations of the efficacy of targeted molecular therapies have triggered the search for additional molecules capable of improving patient survival.Some researchers have confirmed that tumorigenesis and metastasis are frequently associated with altered expression of oligosaccharides on cell surface glycoproteins and glycolipids.Sialic acid is a kind of negatively charged monosaccharides,under the influence of saliva acyltransferase,sialic acid is transferred to galactose which is at the end of glycoprotein or glycolipid through glyosidic bond.More than twenty members of the mammalian sialyltransferase family have been identified to date,divided into four subfamilies according to the carbohydrate linkage synthesized: ?-galactoside?2,3-sialyltransferases(ST3Gal I–VI);?-galactoside ?2,6-sialyltransferases(ST6Gal-I and II);Gal NAc ?2,6-sialyltransferases(ST6Gal NAc I–VI);and ?2,8-sialyltransferases(ST8Sia I–VI).ST6Gal-I catalyzes the sialic acid attached to outermost Gal of Gal?1-4Glc NAc disaccharide units of N-glycan with a ?2-6-glycosidic bond.Thedifferent sialic acid levels on different tumor cells,resulting from elevated ST6Gal-I expression and activity,have been detected in tumor cells,and often correlates with cancer progression,metastasis,and poor prognosis.The structure of sialic acid have changed,which associate with the development of cancer,including the change of sialyl Tn,sialyl Lewis antigen(s Le),?2,6 sialic acid lactose and ganglioside.Abnormal glycosylation results from glycosyltransferase dysregulation,which increasing evidence indicates that is a key phenomenon in many malignancies,including cancers of the breast,colon,and ovary,and leukemia and renal carcinomas.Our original study found that ST6Gal-I had positively associated with malignant phenotype of mouse hepatoma cells,but the mechanisms and mediators for these functions are largely uncovered,especially in hepatocellular carcinoma.Objective: 1.To investigate the clinic pathological relationship of ST6Gal-I in HCC and the relation of ST6Gal-I and HCC cancer patients' survival;2.To obtain the expression levels and location of ST6Gal-I in different human hepatocellular carcinoma cell lines,then screening for the overexpression of ST6Gal-I in Huh-7 monoclonal cell line;3.To determine the influence of overexpression or knockdown of ST6Gal-I on the proliferation,migration and invasion of Huh-7/MHCC97-H cells in vitro;4.To explore the expression levels of ST6Gal-I over the course of DENA-induced liver tumorigenesis and the affection on the growth rates of subcutaneous tumor when knockdown the expression of ST6Gal-I in vivo;5.To examine the molecular mechanism of ST6Gal-I on the proliferation,migration and invasion in Huh-7/MHCC97-H cells.Methods: 1.Immunohistochemical(IHC)staining was used to investigate the clinic pathological relationship of ST6Gal-I in HCC;2.Pearson's chi-squared and Kaplan–Meier test the relation of ST6Gal-I and HCC cancer patients' survival;3.The expression level and location of ST6Gal-I in different human hepatocellular carcinoma cell lines obtained by immunofluorescence;4.The pc DNA3.1/ST6Gal-I plasmid was transfected into Huh-7 cell lines using lipofectamine reagent,and the Huh-7/ST6Gal-I monoclonal cell line was screened by G418 selection and limited dilution.q PCR,Western Blot,immunofluorescence and flow cytometry(FCM)were performed toexamine the expression of ST6Gal-I in Huh-7/ST6Gal-I monoclonal cell line;5.Soft agar colony formation,cck-8,flow cytometry,scratch assay and transwell assays were explored the influence of overexpression or knockdown of ST6Gal-I on the proliferation,invasion and migration abilities in Huh-7/MHCC97-H cell lines in vitro;6.Tumorigenicity in athymic nude mice was applied to examine the effect of ST6Gal-I on the cell proliferation in vivo.7.Western blot and IHC were used to explore the expression levels of ST6Gal-I during the course of DEN/CCl4-induced liver tumorigenesis;8.The protein expression levels of Wnt/?-catenin signaling pathway were analyzed by western blot and immunofluorescence in ST6Gal-I up-/down-regulation cell lines.Results: 1.Compared with non-tumor liver sections,ST6Gal-I highly expressed in HCC tissues;2.Overexpression of ST6Gal-I notably correlated with patients' sex,number of lesions and pathologic grade,and predicts poor prognosis in HCC patients;3.In Golgi apparatus,the highest level of ST6Gal-I was observed in MHCC97-H cells,while ST6Gal-I exhibited low expression in Huh-7 cells;4.ST6Gal-I overexpression/knockdown promotes/attenuates Huh-7/MHCC97-H cell proliferation,invasion and migration in vitro;5.Growth rate,tumor size and weight were dramatically decreased in the MHCC97-H/sh ST6Gal-I group compared with the control groups(MHCC97-H and Negative control cell lines);6.ST6Gal-I expression levels increased significantly over the course of DENA-induced liver tumorigenesis;7.ST6Gal-I overexpression/knockdown significantly increased/restrained the expression levels of ?-catenin,p-GSK-3?,c-Myc,TCF1,TCF4,Cyclin D1 in Huh-7/MHCC97-H cell lines.Conclusion: 1.ST6Gal-I highly expression in high malignant stage HCC tissues,and the positive incidence of ST6Gal-I significantly correlated with patients' sex,number of lesions and pathologic grade.Moreover upregulation ST6Gal-I was correlated with poor prognosis;2.Stable upregulation of ST6Gal-I in Huh-7monoclonal cell line were successfully obtained and MHCC97-H/sh ST6Gal-I cell line was kept in laboratory,the two cell lines were used to explored the abilities of ST6Gal-I up-/down-regulation could promote/reduce the proliferation,invasion and migratory in HCC cells by in vitro and in vivo;3.Upregulation/knockdown the expression of ST6Gal-I could augment/inhibit Wnt/?-catenin signaling pathway by urging/blocking?-catenin entry into the nucleus to influence the HCC development.