The Study Of N-glycans In CRC

Objective: Aberrant glycosylation is highly related with the development of various cancers.The aberrant N-glycan in tumor tissues can be as tumor diagnosis markers.The aim of this study was to analyze the aberrant N-glycans in CRC,and identify the structure of aberrant N-glycans in detail with glycomics method.Find out new targets for the diagnosis and therapy of CRC and provide the structure basis for the study about the function of aberrant N-glycans in CRC.Methods:(1)Total proteins were extracted from CRC tumor tissues and pertumortal tissues,salt ions were removed by WM10000 dialysis.(2)N-glycans were released by PNGaseF.Total N-glycans were purified by Sep-Pak C18 cartridges and porous graphitized carbon(PGC)column and reduced by NaBH4.(3)The purified and reduced N-glycans were methylated with methyl iodide and extracted to remove impurities.(4)Permethylated N-glycans of the glycoproteins from 43 colorectal cancer patients(including CRC tissues and pertumortal tissues)were detected using LTQ-ESI-MS which appeared to be a highly sensitive and informative approach to detect and identify N-glycans,in order to further confirm the structure of the N-glcans,we used sequential multistage MS technology to compare their linkage and branching spectra details.(5)various statistical methods were used,including t test,partial least squares discriminant analysis(PLS-DA)and receiver operating characteristic curve(ROC)to evaluate the MS profiles.Results: The expression of 11 N-glycans was significantly(P<0.05)different between colorectal cancer tissues and controls.There were 9 N-glycans including M/Z973,1060,1075,1162,1177,1264,1352,1055 and 1279 were lower in colorectal cancer tissues than in pertumoral tissues and 2 N-glycans including M/Z 1116 and 1228 were higher in colorectal cancer tissues than controls.We further analyzed the structure of the 11 N-glycans in detail finding that the M/Z 973,1060,1075,1162,1177,1264,1352 were bisected N-glycans and M/Z 1055,1279 were two antennae and three antennae complex N-glycans.On the contrary,M/Z 1116 and 1228 were higher incolorectal cancer tissues than controls.The ion of M/Z 1116 was a high mannose N-glycan and the ion of M/Z 1228 was bifucosylated N-glycan.To further analyzed if it existed diagnostic potential,ROC analysis was used.Receiver operating characteristic(ROC)analysis of bisected N-glycans(M/Z 973,1177,1264,sum of the three peak ions and bisected N-glycans),comparing the colorectal cancer tissues and pertumoral tissues.The AUC of bisected N-glycans was 0.814.These results suggest that the N-glycans containing bisecting GlcNAc structure may serve as a potential marker for colorectal cancer.Interestingly,we observed that the bisected N-glycans were declined with the stage increased.Through age-related analysis,found that there was no relationship between the bisecting structure as clinical diagnosis marker with patients' age(P>0.05).We didn't find bisecting structure N-glycans changed in esophageal carcinoma tissues.Conclusion: colorectal cancer tissues were collected,N-glycans were extracted and analyzed by LTQ-ESI-MS.We found so many aberrant N-glycans from colorectal cancer tissues.Bisected N-glycans were decreased significantly,M/Z 1055 and M/Z1279 were two antennae and three antennae complex N-glycans.On the contrary,M/Z1116 and M/Z 1228 were high in colorectal cancer tissues than controls.The AUC of bisected N-glycans had a higher diagnosis specificity and sensitivity and in the digestive of esophageal cancer was not found the same phenomenon,this phenomenon illustrate the bisected N-glycans maybe colorectal cancer specific expression.The decline of bisected N-glycans was related with stage of CRC significantly.We observed that the bisected N-glycans were declined with the stage increased.