Identification And Proteomic Analysis Of Serum Exosomes Extracted From Patients Of Giant Cell Tumor Of Bone

?Objective?As traditional diagnostic method of needle biopsy for giant tumor of bone(GCT)has inherent drawbacks of invasiveness and high false probability,it is necessary to develop a novel diagnosing approach.Exosomes,as one of the liquid biopsies,tend to be a potential diagnosing technique in the near future.This study aimed to use mass spectrometry proteomic strategy to identify exosomal proteins derived from serum of patients with GCT and apply bioinformatics to analyze their relevant features and function ?Methods and materials?Serum exosomes of patients with GCT and non-tumors were isolated and purified through the exoEasy reagent method.The techniques of western blotting,NanoSight particle counter analysis and transmission electron microscope(TEM)were used to identify the extracted exosomes.Major proteins from serum exosomes were identified by label free analysis followed with screening for differentially expressed proteins via two different quantitative methods of LFQ and iBAQ respectively.The common expressed protein in the two groups were analyzed by Gene Set Enrichment Analysis(GSEA)comprised of pathway enrichment analysis based on Reactome database and GO classification analysis.The interaction of network protein was analyzed by String system.?Results?The typical saucer-like and oval shape of serum exosomes could be observed by TEM.The average diameter of exosome granules was 223.2 nm with the average of 200.4nm,of which 24.8% were between 30 nm and 150 nm in diameter.It was confirmed by the western blot that the expression of CD9 and CD63,surface marker of exosomes,could be detected.Through protein sequencing analysis,we detected a total of 412 proteins in serum exosomes of giant cell tumors and non-tumor patients.Of those,82 proteins were identified with significantly different expression.Among these differentially expressed proteins,45 of them were expressed only in the group of giant cell tumors of the bone,30 were expressed only in the non-tumor group,and the remaining 7 proteins were expressed in both groups.Given the small number of co-expressed proteins with significant difference,all proteins identified in both groups were analyzed by GSEA to find the difference between GCT and non-tumor group.Firstly,the protein set was enriched into the Reactome pathway database,and only one pathway gene set was obtained,which was highly expressed in the GCT group with no significant difference(p value = 0.90);Then the protein set was enriched into GO.In the database,a total of 71 gene sets were enriched,including 67 functional gene sets that were up-regulated in the GCT group and four functional gene sets that were up-regulated in the non-tumor group.It's worth noting that homeostatic process(biological process)and lipid binding(molecular function)were significantly up-regulated in the GCT group.In addition,the GSEA analysis was also performed on the protein quantified by the iBAQ method.There are three Reactome pathway gene sets obtained,of which two were up-regulated in the GCT group and one was up-regulated in the non-tumor group;on the other way 143 gene sets were enriched but with no significant difference in the GO database,of which 89 were actually up-regulated in the GCT group and 54 were up-regulated in the non-tumor group.For those significantly enriched to GO,the core gene of the function is screened out,and the gene interaction network of the core gene is constructed.Through the network,it was recognized that the APOA2 gene was found in both of the two enriched GO function and had a strong interaction with other core genes.?Conclusion?Compared with characteristics of exosomal proteomics in patients with non-tumors,2 signaling pathways relate genes,homeostatic process and lipid binding were expressed with significant difference in patients with GCT.The 2 signaling pathways and the role of node proteins in the interaction among different proteins would be discussed in our future research.