Study On Glycopatterns Of Glycoprotein And Glycosphingolipid In Glioma Cell Lines

Background:Glioma is a common primary intracranial tumor with a relatively low incidence but a very high mortality rate,the five-year survival rate for some types of glioma is less than 5%even after treatment.In view of the increasingly severe treatment status of glioma,the detection and treatment still follow the traditional imaging detection and surgical chemoradiotherapy,this leads to generally poor prognosis.With the development of molecular biology tools,the research on glioma at the micro level has made great progress,but the existing progress focuses more on the function of a single protein or DNA.In this study,glycoprotein and glycosphingolipid isolated from glioma cells were used as samples,the lectin microarray was used to identify total protein and membrane protein and glycosphingolipid glycan spectrum of glioma cells.Analysis of abnormal expression of glycan,and screening of potential markers were conducted by comparing the data with glycopartterns from normal glial cells.Finally,it laid a foundation for the discovery of glycan markers in glioma cells.Method:This study used two glioblastoma cells U87MG and A172,two astrocytoma cells SW1088 and CCD-STTG1,one immortal fetal glial cell SVGp12 as experimental samples.The total protein,membrane protein and glycosphingolipid were extracted after cell culture.The obtained samples were fluorescently labeled and incubated with lectin microarray,after that the microarray image was scanned and the fluorescence signal was read by GenePix7.0 software.the median-normalization and Raito value analysis of the original data are carried out then.Finally,compare the different of glycoproteins and glycosphinolipids glycan spectrum in glioma cells,the glycan structures with significantly different expression were screened.Result:15 lectins with a significant overall up/down-regulation trend were screened in the comparison of total protein glycan spectrum(Ratio?1.5 or Ratio?0.67,p?0.05).Identify the ECA of Gal?1-3/4GlcNAc,WFA of GalNAc?/?1-3/6Gal,MAL-? of Sia?2-3Gal and Sia?2-3GalNAc,PHA-E of Bisecting GlcNAc,PTL-I of GalNAc,GalNAc?1-3Gal and GalNAc?1-3 Gal?1-3/4Glc,PNA of Gal?1-3GalNAc-Ser/Ter(T),EEL of Gal?1-3(Fuc?1-2)Gal,LTL of Fuc?1-2Gal?1-4GlcNAc and Fuc?1-3(Gal?1-4)GlcNAc,GSL-I of ?GalNAc and ?Gal,VVA of GalNAc and GalNAc?1-3 Gal-Ser/Ter(Tn),MAL-I of Gal? 1-3/4GlcNAc showed a significant up-regulating trend.And identify the NPA of High-Mannose and Man?1-6Man,PWM of Branched(LacNAc)n,GNA of High-Mannose and Man?1-3Man,BPL of Gal?1-3GalNAc and Terminal GalNAc showed a significant down-regulating trend.6 lectins with a significant overall up/down-regulation trend were screened in the comparison of membrane protein glycan spectrum.the glycan identified by PNA and VVA were significantly up-regulated,while the glycan identified by LTL and PTL-I,(GlcNAc)n identified by LEL,Sia?2-6Gal/GalNAc identified by SNA showed a significant down-regulating trend.The glycan identified by the same lectin may not be identical in glycosphingolipids and glycoproteins because of the structure differences between them.In the comparison of glycosphingolipid glycan spectrum,10 lectins with a significant overall up-regulated/down-regulated trend were screened.Identify the EEL of Gal?1-3(Fuc?1-2)Gal,MAL-I of Gal?1-4GlcNAc showed a significant up-regulating trend.And identify the NPA of Fuc?1-4GlcNAc and poly-Man,GNA of ?1-3Man and Galal-3Gal?1-4GlcNAc,MPL of Gal?1-3GalNAc,Gal?1-3GlcNAc and GalNAc,HHL of ?1-3/1-6Man and Galal-3Gal?1-4GlcNAc,LCA of Fuc?1-6GlcNAc,PSA of Fuc?1-6GlcNAc and Fuc?1-3GlcNAc showed a significant down-regulating trend.From the above findings,Gal?1-3/4GlcNAc,Sia?2-3Gal/GalNAc,Bisecting GlcNAc,Fuc?1-3(Gal?1-4)GlcNAc,GalNAc?1-3Gal?1-3/4Glc,GalNAc-Gal and Gala1-3(Fuc?1-2)Gal showed significant up-regulation,High-Mannose and Branched(LacNAc)n showed significant down-regulation in glioma cell total proteins.In the glioma membrane proteins,Gal-GalNAc showed significant up-regulation,GalNAc?1-3 Gal?1-3/4Glc,Fuc?1-3(Gal?1-4)GlcNAc,(GlcNAc)n and Sia?2-6Gal/GalNAc showed significant down-regulation.In the samples of glycosphingolipids,Gal?1-3(Fuc?1-2)Gal and Gal?1-4GlcNAc showed significant up-regulation,Gal?1-3Gal?1-4GlcNAc,Fuc-GlcNAc and Gal-GlcNac showed significant down-regulation.Notably,Sia2-6Gal/GalNAc showed significant down-regulation(p?0.01)in the membrane proteins,and showed down-regulation but not significant in the total proteins(p?0.05).In addition,the glycans with GalNAc-Gal structure showed a significant up-regulation between the total proteins and the membrane proteins,while the glycan Fuc?1-3(Gal?1-4)GlcNAc and GalNAc?1-3Gal?1-3/4Glc showed a significant up-regulation in the total protein,but a significant down-regulation in the membrane protein.These findings suggest that the glycoproteins with GalNAc-Gal glycan in glioma cells may be up-regulated throughout the glioma cell system.The glycoproteins with structure of Fuc?1-3(Gal?1-4)GlcNAc and GalNAc?1-3Gal?1-3/4Glc were up-regulated in the whole glioma cell,but down-regulated on the cell membrane surface.