Research On Glycosylation Of Prostate Specific Antigen In Prostate Cancer

Background Prostate Cancer(PCa)is the most commonly diagnosed cancer and second leading cause of cancer death in man.With the aging of population and development of medical testing technology,the incidence of prostate cancer tends to increase in in China.Prostate Specific Antigen(PSA)was taken into use in clinic in 1990 s,which has significantly increase the early diagnosis efficiency of prostate cancer.However,serum PSA elevation is not cancer specific.Its limited sensitivity has led to overdiagnosis and over treatment of prostate cancer.New biomarkers are needed to improve the early diagnosis of PCa in clinic.In hunman body,about over 50% of protein was glycosylated,these protein were also called glycoproteins.In cancer patients,glycosylation alternation is a common phenomenon.PSA is also one of glycoproteins.But there is only one N-glycosylation site,Asn-69.It was reported that several N-glycosylation of PSA can help discriminate PCa from non-cancers.But almost all the researchers only focused on the aberrant glycosylation in cell lines or protein pools,and other results were found out with few clinic patients.These results could not help increase the diagnostic efficiency.Objective In order to discover aberrant glycosylation in PSA and then evaluate its diagnostic efficiency in clinic,we collect the expressed prostate secretion(EPS)urine of those who received prostate biopsy treatment during Jun.2014 to Oct.2016 in Changhai Hospital,Shanghai,China.In this study,we want to identify out the type of N-glycosylation of PSA and then discriminate prostate cancer from non-cancers according to their alteration in glycosylation of PSA.Method We collect EPS-urine sample of patients who received prostate biopsy treatment in Changhai Hospital,Shanghai,China,during Jun.2015 to Oct.2016 and random choose 58 samples(29 prostate cancers and 29 non-cancers).The associated patient information such as age,PSA level,pathological diagnosis and gleason score were also recorded.According to the pathological result,urine samples were divided into prostate cancer group and benign prostate hyperplasia group.Immunoprecipitation method was applied to purify PSA.After SDS-PAGE,cut off the PSA band.N-glycan was released by PNGase F by the method of in-gel digestion.Then the free N-glycan was labelled with Rapi Fluor-MS kit.The labelled N-glycan sample was purified again with Solid Phase extraction Cartridges.Labelled N-glycan was tested by the UPLC/FLD/ESI-QTOF-MS system.The data should be matched to the glycol database of NIBRT and Glyco Workbench.As a sequence,about 28 different types of N-glycan were identified out.Finally,the ?2-3,6,8,9 Neuraminidase A was used to cut off the sialic acid,and then the digested N-glycan was tested again by the UPLC/FLD/ESI-QTOF-MS system.Compare two sets of data to verify the identification accuracy.Confirming the accurate identification result,the quantitative data of N-glycosylation between cancer and non-cancer group were then analyzed by statistic method.Result Twenty-eight different N-glycan types were at last identified out.Most of them were biantennary complex type glycan.There were three major patterns in PSA glycan profiles.Significance was found in the N-glycan FA2 and FM5A2G2S1 between PCa group and BPH group.Combining FA2,FM5A2G2S1 and PSA,the prediction data can discriminate PCa more efficiently from non-cancers.FA2 was also significantly increased in PCa patients whose Gleason Score was more than 8.Conclusion There were significance in N-glycosylation of PSA.And several glycans of PSA had the potential to be a new biomarker.