Screening And Validation Of The Diagnostic Biomarkers For Non-Small Cell Lung Cancer Based On Blood

Backgrounds: Non-small cell lung cancer(NSCLC)is the most common type that accounts for about 80% of all lung cancer.Due to the high degree of malignancy and rapid progression of lung cancer,more than 70% patients were diagnosed at an advanced stage(stage III/IV).Despite continuous improvements in tissue biopsy and imageology examination,their five-year survival rate is still less than 15%.Therefore,there is an urgent need for a sensitive,minimally invasive indicator for early detection and prognosis to help clinically detect disease early or provide more aggressive treatment to effectively control disease.Exosomes are microvesicle secreted by cells,which play an important role in the growth,metastasis and drug resistance of tumors and the changes in tumor microenvironment.Serum exosomes have been shown to be useful as markers for the diagnosis and metastasis of a variety of cancers.White blood cells constitute the body's first-line immune defense system.The continuous interactions between the white blood cells and the entire body may trigger the specific changes in the gene expression of white blood cells.So observing changes in gene expression in white blood cells can provide a more effective and minimally invasive method for diagnosing and monitoring diseases.Objective: We aimed to screen the differentially expression of serum exosomal proteins and differential expression profile of white blood cell mRNA,and validate their diagnostic values in patients with NSCLC.Methods: Proteomic techniques were used to screen differentially expressed proteins in serum exosomes of healthy volunteers and NSCLC patients,and ?-2-HS-glycoprotein(AHSG)and extracellular matrix protein 1(ECM1)AHSG were screened based on fold changes.Western blot and ELISA were used to verify the expression levels of the two differential proteins in the healthy and NSCLC groups,and the ROC curve was drawn to calculate the diagnostic efficacy of the two proteins as diagnostic markers.At the same time,RNA-seq technology were used to screen differentially expressed mRNA in white blood cells of healthy volunteers and NSCLC patients.Two up-regulated genes(GPX1,PRELID1)were screened according to fold changes,three down-regulated genes(MAP3K7CL,BCL9 L,PCSK7)were selected.Real-time quantitative PCR(RT-PCR)was used to verify the expression levels of five differential mRNAs in the healthy and NSCLC groups,and the ROC curve was drawn to determine the diagnostic efficacy.Statistical analysis were performed using Prism and SPSS by Mann-Whitney nonparametric test,Kruskal-Wallis test and one-way ANOVA.Results: Serum exosomal differential proteins were validated in 46 healthy individuals and 125 NSCLC patients.Compared with the healthy group,the expression levels of AHSG and ECM1 protein in serum exosomes of NSCLCpatients were significantly increased,the diagnostic efficacy of AHSG was 0.736(P < 0.0001),and the diagnostic efficacy of ECM1 was 0.683(P < 0.001).For early stage NSCLC,the diagnostic power of AHSG was 0.682(P < 0.01),and the diagnostic power of ECM1 was 0.656(P < 0.05).When AHSG was combined with ECM1,the diagnostic efficacy of NSCLC patients and early stage NSCLC patients was 0.795 and 0.739,respectively.AHSG,ECM1 combined with the traditional tumor marker CEA significantly improved the diagnostic ability of NSCLC,AUC was 0.938,and the AUC of patients with early satge NSCLC was 0.911.At the same time,144 healthy volunteers and 159 NSCLC patients were enrolled to verify the differential mRNA expression profile of white blood cells.RT-PCR results showed that the expression levels of the five genes were significantly different between NSCLC and healthy subjects(P < 0.001).When the five genes were combined to diagnose NSCLC,the diagnostic efficacy was 0.843,the sensitivity was 74.2%,and the specificity was 79.9%.PRELID1 and PCSK7 genes could be distinguished between healthy group and early stage NSCLC patients(P < 0.05).BCL9 L and PCSK7 were associated with lymph node metastasis(P = 0.0008,P = 0.0212)and PRELID1 was associated with distant metastasis(P = 0.0408).In addition,the down-regulated gene BCL9 L is associated with chemotherapeutic effect.Conclusions: Expression differently of serum exosomal protein AHSG and ECM1 and white blood cells mRNA suggest that they can be used as a potential diagnostic biomarker for NSCLC.Remarkably,our study provided the basis for further research on biology and function.At the same time,it also provides new ideas for the diagnosis of other tumors.