The Role Of Glycosyltransferases β3GnT8and β3GnT2Play On The Cell Signal Transduction Pathway During CD147Inducing MMPs Production

Objective: To investigate the role of glycosyltransferases（β3GnT8andβ3GnT2）play on the cell signal transduction pathway during CD147inducing MMPs production；To investigate the expression of polylactosamine and extracellular matrixmetalloproteinase inducer （EMMPRIN, CD147） in gastric cancer and Glioma tissues,To investigate the Clinical significance of polylactosamine and CD147;To investigatethe effect of⁶⁰Co γ-ray on the expression of glycosyltransferases and carbohydratechains in tumor cell line.Methods:（1） β3GnT8RNA interference vector pSilenCircle-T8Si、control vectorpSilenCircle-T8Scr、β3GnT8sense vector pEGFP-C1-T8S、β3GnT2sense vectorpEGFP-C1-T2S and empty vector pEGFP-C1were transfected stably into U251、LN229and SGC-7901cells by liprofectamine2000. And the stably transfected cell lines weresieved by G418.（2） The GFP fluorescence protein expressed in U251、LN229andSGC-7901cells transfected with sense vector and empty vector groups were observedwith fluorescence microscope. Using RT-PCR and western blotting to measure theβ3GnT8and β3GnT2mRNA and protein expression levels.（3） Flow cytometry andimmunofluorescent staining analysis were utilized to evaluate the expression ofpolylactosamine on the stably transfected cell lines.（4）Using western blotting analysisrevealed the expression of CD147in tumor cell lines and the expression on proteinlevels of HG-CD147by tunicamycin、glycosyltransferases and3’-azidothymidine.（5）RT-PCR analysis was utilized to evaluate the expression of MMP-2by tunicamycin;Flow cytometric analysis was used to detect Cell Cycle by tunicamycin.（6） Analysis ofprotein–protein interaction （PPI） networks of CD147and MMPs and protein CD147and MMPs3D Structure Model.（7）Manufacturing tumor tissue chip, Detecting theexpression of polylactosamine and CD147in tumor tissue chip.（8）Using⁶⁰Co γ-ray to irradiate tumor cell line, Using western blotting to measure the β3GnT8proteinexpression level. Using flow cytometry to detect the effect of⁶⁰Co γ-ray on theexpression of polylactosamine.Results:（1） Vectors were transfected into cells successfully which were observedthrough fluorescence microscope,RT-PCR,western blotting and laser confocalmicroscope. And we got the stably transfected cell lines.（2） Flow cytometry andImmunofluorescent staining experiment indicated that the expression ofpolylactosamine increased when increasing the expression of β3GnT8and theexpression of polylactosamine decreased when decreasing the expression of β3GnT8.（3）The western blotting analysis revealed:①CD147did have two different forms,high glycosylated （HG） form and low glycosylated （LG） in U251and SGC-7901;②After tunicamycin treatment in U251and SGC-7901, a single newband of molecularweight consistent with the size of the core protein was visualized;③LN229cells andSGC-7901cells transfected with β3GnT8siRNA exhibited a directly reduction at thelevels of HG-CD147protein,compared with β3GnT8Scr transfectants and NC；④AfterAZT treatment in SGC-7901, The fraction of HG-CD147remaining at45-65kDadecreased gradually, comparing with HG-CD147/control.（4） After tunicamycintreatment in SGC-7901and U251, the level of MMP-2almost disappeared, comparingwith MMP-2/control（.5）Tumicamycin treatment changed the cell cycle distributions ofSGC-7901cells.Tumicamycin-treated cells increased the percentage of the cells in theG1phase in a dose-dependent manner.（6）The analysis of protein–protein interaction（PPI） networks of CD147and MMPs was derived from an analysis of the STRINGdatabase.The results reveal that CD147has related signal pathway with MMPsincluding MMP-1、MMP-2、MMP-3、MMP-7、MMP-9、MMP-11and MMP-14. wefound three N-linked （GlcNAc...） glycosylation sites（Asn160、Asn268、Asn302） ofCD147in STRING database.（7）Manufacturing gastric carcinoma tissue、adjacentnon-neoplastic gastric tissue and human glioma tissue chip Successfully.（8）Theexpression of polylactosamine in gastric cancer is more than that in normal gastricmucosa epithelial cells, The expression of polylactosamine in diffuse type gastric canceris more than that in intestinal type gastric cancer. The expression of polylactosamine isalso strong in human glioma tissue microarray.（9）CD147was detected in gastric cancer cell cytoplasm and membrane, little of CD147was detected in normal gastricmucosa epithelial cells.CD147is also strong in human glioma tissue microarray.（10）The expression of β3GnT8using west blotting to detect and polylactosamine usingflow cytometry to detect decreased gradually as the dose of⁶⁰Co γ-ray increased.Conclusion:（1） Established the stable upregulation and downregulation of3GnT8andβ3GnT2cell lines.（2） β3GnT8is involved in the biosynthesis of polylactosaminechains on the β1,6-branch of N-glycan.（3）After tunicamycin treatment in U251andSGC-7901, a single newband of molecular weight consistent with the size of the coreprotein was visualized by Western blotting. These results suggested N-linkedglycosylation of CD147was highly sensitive to inhibition by tunicamycin.（4）β3GnT8and β3GnT2were involved in the biosynthesis of polylactosamine chains of the HGform of CD147. β3GnT8and β3GnT2regulated the expression of HG-CD147onprotein level in LN229and SGC-7901cells.（5）After cells were treated with AZT, thefraction of HG-CD147decreased gradually, compared with HG-CD147/control. AsHG-CD147determines MMP expression, AZT maybe affect MMP expression.MMPsare one class of molecules that have been shown to play an integral role in tumorprogression,facilitating cell migration and tumor invasion through their ability todegrade ECM. So we can conclude that not only3′-AZT is significant to treat HIVpositive patients,but also it may be an antineoplastic drug used in the treatment ofcertain cancers.（6） Tunicamycin affects the expression of MMP-2, Tumicamycin-treated cells increased the percentage of the cells in the G1phase in a dose-dependentmanner.（7）CD147and MMPs reveal that CD147has related signal pathway withMMPs including MMP-1、MMP-2、MMP-3、MMP-7、MMP-9、MMP-11and MMP-14.（8）Polylactosamine and CD147expressed much in gastric cancer tissue and humanglioma tissue.（9）The expression of β3GnT8and polylactosamine was relativelysensitive to⁶⁰Co γ-ray, The expression of β3GnT8and polylactosamine was negativecorrelation to the dose of⁶⁰Co γ-ray.