| BACKGROUND:Breast cancer?BC?is a common malignant tumor in women and the incidence and mortality rates of BC is highest among malignant tumors in women by now.It is necessary to study the diagnostic methods of early-stage breast cancer because the incidence of breast cancer has shown a younger trend in recent years.The diagnosis of breast cancer is mostly based on pathological biopsy and assisted by molybdenum target technology,but these diagnostic methods could cause different level of pain to patients.With the development of tumors,the researches on saliva as a diagnostic sample are increased gradually.In our laboratory,we've analyzed the abnormal expression of glycans in the saliva samples of 217 patients with early-stage breast cancer using Lectin Microarray and established a mathematical model.The results showed that lectin BS-I had the highest sensitivity and specificity in the diagnosis of early-stage breast cancer?AUC:0.763,sensitivity:0.778,and specificity:0.631?.In this study,we used matrix-assisted laser desorption ionization time-of-flight mass spectrometry?MALDI-TOF/TOF-MS?to further study the galactosylated N-/O-glycans which were specifically recognized by lectin BS-I.These discoveries could be used theoretically for anti-cancer drugs screening,early diagnosis and treatment of breast cancer.METHODS:We compared the expression of BS-I-recognized salivary protein glycans in healthy volunteers?HV?,benign breast cyst or tumour patients?BB?and early-stage breast cancer patients?BC-I/BC-II?.Firstly,we used saliva microarray to detect the differently expressed glycans that identified by lectin BS-I in four saliva groups.Secondly,the salivary protein was purified by BS-I coupled magnetic particles.N-glycan and O-glycan on glycoproteins were separated by PNGase F digestion and NaClO oxidation respectively.Thirdly,the separated glycoproteins were analyzed by MALDI-TOF/TOF-MS.RESULTS:Statistically,the glycans that recognized by lectin BS-I in saliva chip were significantly different between HV and BC-I group?Mean rank difference=2.00,P=0.008?,HV and BC-II group?Mean rank difference=2.21,P<0.001?,BB and BC-I group?Mean rank difference=1.51,P=0.044?,BB and BC-II group?Mean rank difference=1.67,P=0.008?,while there were no significant differences between HV and BB group?Mean ran difference=1.32,P=0.290?and BC-I with BC-II?Mean rank difference=1.10,P=0.724?.The results of mass spectrometry showed that 12/12,12/22,8/26,9/12 N-/O-glycans were identified in the saliva of HV,BB,BC-I and BC-II groups.Among them,11/10,10/19,7/24,7/9 N-/O-glycans had?-Gal,?-GalNAc,Gal?1-3Gal and Gal?1-6Glc structures that were specifically identified by BS-I.In the identified glycans,only 5 N-glycans?e.g.m/z1753.945?NeuAc?1?GlcNAc?1?GalNAc?1+?Man?3?GlcNAc?2?Fuc?1,1767.975?Gal?2?Man?1?GlcNAc?1+?Man?3?GlcNAc?2?Fuc?1,2080.155?Fuc?2?GlcNAc?2?GalNAc?2+?Man?3?GlcNAc?2?Fuc?1?were found only in HV group.There were only 2 N-glycans?m/z3065.498?NeuAc?2?Fuc?2?Gal?4?GlcNAc?3+?Man?3?GlcNAc?2?,3539.750?NeuAc?1?Gal?6?GlcNAc?6+?Man?3?GlcNAc?2?Fuc?1)and 2 O-glycans?m/z 734.165?Fuc?2?Gal?1?GalNAc?1,896.124?Fuc?2?Gal?2?GalNAc?1?were found only in BB group.There were 1 N-glycans?m/z 2773.977?NeuAc?1?Fuc?1?Gal?4?GlcNAc?3+?Man?3?GlcNAc?2?Fuc?1?and 7 O-glycans?e.g.m/z 868.295?Fuc?1?GlcNAc?1?GalNAc?2,882.243?Fuc?1?Gal?1?Glc?1,884.270?NeuAc?2?GalNAc?1?were found in BC-I group.3 N-glycans?m/z2101.805?Fuc?2?Gal?2?GlcNAc?2+?Man?3?GlcNAc?2?Fuc?1,2246.870?NeuAc?1?Fuc?1?Gal?2?GlcNAc?2+?Man?3?GlcNAc?2?Fuc?1,2392.909?NeuAc?1?Fuc?3?Gal?2?GlcNAc?2+?Man?3?GlcNAc?2?were only found in BC-II group.These findings may contribute to the study of biomarkers of breast cancer based on changes in galactosylated N/O-glycans in saliva. |
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