Application Of Exosomal Protein And MiRNAs In The Metastasis Prediction Of NSCLC

Backgrounds: Non-small cell lung cancer(NSCLC),the predominant category of lung cancer,accounts for ~85% of all cases.Despite improvements in traditional treatments,including surgery,followed by radiotherapy and/or chemotherapy,the prognosis of NSCLC remains poor,with a 5-year overall survival rate of 10~15%.Thus,there is an urgent need for a sensitive biomarker to provides an objective basis for the early diagnosis,early treatment and prognosis of metastatic NSCLC.Tumor-derived exosomes(TDEs)have been demonstrated to promote cancer progression,allowing the distal organ site to be prepared as a premetastatic niche.In addition,exosomal contents,such as RNA and proteins,are protected from proteinase-dependent degradation and thus can be stably detected in the circulating plasma and serum,making them ideal biomarkers for a number of clinical applications.Moreover,TDEs are readily available in blood samples where they constitute potential biomarkers of tumor.Methods: Tandem mass tags combined with multidimensional liquid chromatography and mass spectrometry analysis were used for screening the proteomic profiles of the exosomes.The quantitative proteome,significant pathway,and functional categories of patients with metastatic and nonmetastatic NSCLCs and healthy donors were investigated.According to the differential expression multiple,a differentially expressed protein,lipopolysaccharide-binding proteins(LBP)was screened and verified by Western Blots and ELISA,Receiver operating characteristic(ROC)curve was used to determine the diagnostic efficiency.Meanwhile,miRCURYTM LNA were used to determine distinct miRNA profiles of plasma exosomes in a discovery cohort of healthy donors(n=5),patients with metastatic NSCLC(n=5)and patients with non-metastatic NSCLC(n=5).Three potential candidate miRNAs were selected based on the differential expression profiles.The discovery set data was validated by quantitative real-time PCR(RT-PCR)using a validation cohort,and statistical analysis SPSS22.0 software was used to determine the diagnostic efficiency.Results: In total 552 proteins of the 628 protein groups identified were quantified.Bioinformatic analysis revealed that the quantifiable proteins were mainly involved in multiple biological functions,metastasis-related pathways.Moreover,the LBPs in the exosomes were found to be well distinguished between patients with metastatic and patients with nonmetastatic NSCLC.The area under the curve(AUC)was 0.786 with a sensitivity of 84.5% and specificity of 72.3%(p<0.0001).The circulating LBPs were also well distinguishable between metastatic and nonmetastatic NSCLCs,the AUC was 0.683 with a sensitivity of 79.5% and specificity of 47.2%(p=0.005).At the same time,we screened out some differentially expressed miRNAs(let-7f-5p,miR-320 a,miR-622)by miRCURYTM LNA array.Results demonstrated that the level of let-7f-5p was decreased in plasma exosomes of NSCLC patients(P<0.0001).Further analysis revealed that miR-320 a,miR-622 and let-7f-5p levels could significantly segregate patients with metastatic NSCLC from patients with non-metastatic NSCLC(P<0.0001,P<0.0001 and P=0.023,respectively).In addition,the combination of let-7f-5p and the conventional biomarkers(CEA and Cyfra21-1)generated an AUC of 0.981 for the diagnosis of NSCLC patients,a sensitivity of 94.7% and specificity of 93.3% in identifying patients with NSCLC,which was significantly higher than any single biomarker.And the sensitivity and specificity of the combined four tumor markers(miR-320 a,miR-622,CEA,Cyfra21-1)in diagnosing NSCLC distant metastases were 83.8% and 94.7%,respectively,and the AUC was 0.900.Concludes: Accroding to the LC-MS/MS and miRCURYTM LNA array,patients with metastatic and nonmetastatic NSCLCs differed in exosome-related proteins and miRNAs in the serum.The exosomal LBPs and miRNAs might be promising and effective candidates for the diagnosis of metastatic NSCLC.This study,therefore,also provides a new idea for noninvasive biomarkers for other metastatic cancers,and the clinical application of exosomes will have better prospect,which suggest that TEDs may mediate the metastasis of NSCLC.