Expression Of Serum Exosomes Hsa-miR-17-5p And Hsa-miR-20b-5p In Non-small Cell Lung Cancer And Their Diagnostic Value

Objective:Lung cancer is the world’s second highest incidence and mortality of malignant tumors.Lung cancer is mainly includes non-small cell lung cancer(NSCLC)and small cell lung cancer(NSCLC),of which NSCLC accounts for about 85%of the incidence of lung cancer and is the main type of lung cancer.The onset of NSCLC is insidious,most of the which are not diagnosed until the middle or advanced stage,and the 5-year survival rate is low.Early methods for clinical diagnosis of NSCLC are very limited,with disadvantages such as poor patient compliance,low sensitivity,low specificity,etc.,and the detection of NSCLC is already at an advanced stage,which leads to the missing of the best treatment opportunity.Therefore,it is of Paramount importance to find novel tumor markers for NSCLC with rapid,non-invasive,high specificity and high sensitivity.In recent years,the abnormal expression of various microRNAs(miRNAs)in exosomes of patients with non-small cell lung cancer(NSCLC)has become a research hotspot.More and more studies have confirmed that exosome-coated miRNAs play a key role in the occurrence,development and metastasis of tumors,and can be used as a potential new non-invasive biomarker for the diagnosis of tumors.This study aims to investigate the relative expression of miRNA molecules encoded by serum exosomes from three homologous gene clusters in patients with NSCLC,and to evaluate their clinical diagnostic value for NSCLC,so as to provide more valuable new biomarkers for the diagnosis of NSCLC.Methods:1.First,with "NSCLC,miRNA" as the key words,relevant literature was searched from PubMed database to find the miRNAs differentially expressed in NSCLC patients as the miRNA molecules to be studied.2.Exosomes were isolated and extracted from the sera of NSCLC patients and healthy controls using the ExoQuickTM Solution kit by precipitation method,and exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis,and exosome marker protein TSG101 and CD9 immunoblotting techniques.3.In the screening stage,48 patients with NSCLC and 48 healthy controls were randomly included in the training set,and the relative expression levels of candidate miRNAs in serum exosomes were detected by qRT-PCR.The miRNAs with significant expression differences between the two groups were screened by statistical analysis,and the receiver operating curve(ROC)was plotted.Calculate the area under the curve(AUC).4.In the validation stage,131 patients with NSCLC and 96 healthy controls were randomly selected to be included in the validation set.The relative expression of the differentially expressed miRNAs screened in the training set was further verified in the verification set,and the diagnostic efficacy was evaluated.5.The screened serum exosome miRNA molecules were combined with current clinical tumor markers of non-small cell lung cancer related proteins,including squamous cell carcinoma antigen(SCCA),cytokeratin 19 fragment(CYFRA21-1)and carcinoembryonic antigen(CEA),through Logistic multiple regression analysis.The diagnostic model of serum exosome miRNA in NSCLC was established,and the diagnostic efficacy of the model and its relationship with TNM stage Ⅰ,Ⅱ,Ⅲ tumor stage of NSCLC were evaluated by the area under the ROC curve.6.The correlation between the relative expression levels of serum exosomes hsa-miR-17-5p and hsa-miR-20b-5p in the training set and the clinicopathological characteristics of patients with NSCLC was statistically analyzed and verified respectively.Results:1.By searching PubMed database,the literature reported a total of 15 miRNAs encoded by three homologous miRNA gene clusters,including miR-17-92,miR-106a-363,and miR-106b-25 families,due to their high expression in bronchoalveolar lavage fluid of patients with non-small cell lung cancer.So they’re candidate molecules.2.Exosomes were isolated and extracted from the sera of patients with NSCLC and healthy controls.NTA technology detected 99.5%of particle diameter distributed in the range of 30～200nm,western blotting detected exosomal-expressed proteins CD9 and TSG101,and the complete exosome morphology was observed by transmission electron microscopy.3.In the screen:ing stage,among the 15 candidate miRNAs,hsa-miR-17-5p and hsa-miR-20b-5p were differentially expressed in NSCLC and healthy control sera.The serum exosomes hsa-miR-17-5p and hsa-miR-20b-5p in the training set were both overexpressed in patients with NSCLC(all P<0.0001),and the area under the receiver operating curve(AUC)was 0.834 and 0.808,respectively.4.In the validation stage,compared with healthy controls,serum exosomes hsa-miR-17-5p and hsa-miR-20b-5p were also highly expressed in patients with NSCLC in the validation set(both P<0.0001),and AUC were 0.784 and 0.756,respectively.5.To establish a diagnostic model of non-small cell lung cancer.The AUC of the model for NSCLC in the training set was 0.914(95%CI=0.839-0.961),the sensitivity was 91.6%,and the specificity was 83.3%.The AUC of the model for NSCLC in the validation set was 0.824(95%CI=0.7688-0.871),the sensitivity was 72.5%,and the specificity was 72.9%.The diagnosis model of TNM staging in patients with non-small cell lung cancer Ⅰ,Ⅱ,Ⅲ period diagnosis efficiency of 0.783,0.832 and 0.902 respectively.6.In the validation set,hsa-miR-17-5p was statistically significantly correlated with tumor size,histological type,and TNM stage in patients with NSCLC(P<0.05),there was a statistically significant correlation between hsa-miR-20b-5p and lymph node metastasis(P<0.05).Conclusions:1.The two molecules,hsa-miR-17-5p and hsa-miR-20b-5p,were significantly overexpressed in serum exosomes of patients with NSCLC and had high clinical diagnostic value for NSCLC.2.The serum exosome model based on Logistic regression analysis has a high diagnostic efficiency for NSCLC,which is significantly related to pathological stages and is of great significance for the diagnosis of NSCLC.