Screening And Identification Of Nasopharyngeal Carcinoma Related Serum Marker EFEMP2 And Its Regulatory Pathway

Objective The differential proteins in serum of patients with nasopharyngeal carcinoma were screened by proteomic technique,and their molecular functions and related signal pathways were studied.Screening serum proteomic specific markers of nasopharyngeal carcinoma will provide key target information for the mechanism study of nasopharyngeal carcinoma,and provide a new strategy for the diagnosis,monitoring,and treatment for clinical translational research.Methods In 2020,Plasma samples from 8 normal subjects and 16 nasopharyngeal carcinoma patients（grouped by EBV: 8 patients with EBV+ and 8 patients with EBV-;Group by the degree of metastasis: 8 cases of M0 and 8 cases of M1）were collected in Wuhan University Zhongnan Hospital.And 8 sex-age-matched healthy subjects were included as a control group.DIA technology was used to screen out differentially expressed proteins,and several proteins with the most significant differences were selected as target proteins.In addition,the bioinformatics function prediction results of differential proteins GO and KEGG were used to select key functions and signaling pathways for subsequent research and verification.The serum concentrations of candidate differential proteins in 63 nasopharyngeal carcinoma patients and 20 matched normal subjects were detected by a commercial ELISA kit.DNA was extracted from plasma and PBMC of nasopharyngeal carcinoma patients with a nucleic acid extraction kit,and EBV DNA load was detected with EBV quantitative kit.Results（1）DIA screened 90 differentially expressed proteins（DEPs）from NPC vs Healthy group.Compared with the Healthy group,16 kinds of DEPs were up-regulated and 74 kinds of DEPs were down-regulated.（2）According to more stringent screening criteria:（1）FC value,P-value,and background value met the set criteria,（2）the tissue expression characteristics in the standard public gene chip database were consistent,（3）the target protein expression abundance in serum was high,5 differential proteins（EFEMP2,FBLN5,YWHAE,LSP1,DBH）were selected for serological verification.（3）Determination of target protein: The results of 63 NPC and 20 Healthy serum samples showed that only EFEMP2 and DBH were significantly high in NPC patients’ serum（P<0.05）,and EFEMP2 had a significant diagnostic value as a biomarker of NPC circulation（AUC=0.7,P<0.05）.Therefore,EFEMP2 was selected as the target protein.（4）According to the prediction of bioinformatics function,the interaction between viral proteins and cytokines and cytokine receptors is one of the main signaling pathways involved in up-regulating differential proteins in NPC vs Healthy group.Therefore,we hypothesized that the secretory target protein EFEMP2 affects the NPC tumor immune microenvironment by influencing cytokines secreted by immune cells.Cytokines regulate the secretion of EFEMP2: In vivo,EFEMP2 concentration in serum of NPC patients was significantly positively correlated with IL-4,IL-6,IL-10,and IFN-γ,the most commonly secreted cytokines of PBMC.The correlation coefficients were r=0.274,P=0.03,R =0.260,P=0.039,R =0.466,P=0.000 and R =0.322,P=0.01.In vitro,EFEMP2 concentration in the supernatant of 5 NPC cell lines was significantly higher than that of PBMC of NPC patients,and the correlation between EFEMP2 concentration and IL-6 was most significant after co-culture of NPC cell lines and PBMC of NPC patients（r=0.316,P=0.028）.（6）Cytokines were correlated with EBV viral load in NPC patients: positive plasma EBV+ and EBV viral load were correlated with tumor stage in NPC patients（P<0.05）.Our study showed that some cytokines,such as IL-6 and IL-10,had a good correlation with EBV DNA load（R=0.362,P=0.025 and R=0.497,P=0.001）,suggesting that EBV viral load might influence the possibility of NPC typing and prognosis by regulating cytokines.Conclusions This research through the DIA proteomics technology and clinical samples,high serum protein expression in NPC patients EFEMP2,experiments in vivo and in vitro showed that prompt part PBMC cytokines can influence the expression of serum marker EFEMP2 level,participation in nasopharyngeal carcinoma cell proliferation,apoptosis,protein synthesis,and cycle regulation,and other important physiological processes.The interaction between Epstein-Barr virus and cytokines directly or indirectly affects the development of Epstein-Barr virus-associated NPC.In conclusion,the target protein EFEMP2 and its regulatory pathway were screened out in this study,suggesting an important entry point for the study of tumor immune microenvironment,but further research is needed to clarify its mechanism of action.