The Study Of A Panel Of Serum MiRNAs With Serum Exosomal MiRNAs In Early Diagnosis For Non-small-cell Lung Cancer

Background Non-small-cell lung cancer(NSCLC)is the dominant pathological type of lung cancer.Due to the absence of apparent symptoms at an early stage,most patients with NSCLC miss the best opportunity of surgical treatment,leading to a survival rate of less than15%.MicroRNAs(miRNAs)are a class of small RNAs,which are stable in both serum and exosome.Studies have shown that detection of abnormal miRNA expressions in serum and/or serum exosome is helpful for early diagnosis of NSCLC patients.ObjectivesThis study aims to investigate the value of combined detection of two forms of miRNA in serum for the early diagnosis of NSCLC patients by simultaneous detection of 8common serum miRNAs including miR-21-5p,miR-126-3p,miR-141-3p,miR-146a-5p,miR-155-5p,miR-222-3p,miR-223-3p,miR-486-5p and 8 corresponding serum exosomal miRNAs in early NSCLC patients.And then,mimics and inhibitor for serum exosomal miRNAs associated with the early diagnosis of NSCLC were constructed and transfected into NSCLC cells,respectively.We also intend to further explore the effects of miRNAs overexpression/knockdown on the on the viability,migration,invasion,and apoptosis of NSCLC cells and explain its potential role on the occurrence and development of NSCLC.Methods1.We measured 8 serum miRNAs and corresponding serum exosomal miRNAs including miR-21-5p,miR-126-3p,miR-141-3p,miR-146a-5p,miR-155-5p,miR-222-3p,miR-223-3p,and miR-486-5p in 48 patients with early NSCLC at Ⅰ/Ⅱ stage,32 patients with lung benign lesion(LBL),and 48 healthy control(HC)by q RT-PCR.The receiver operating characteristic curve(ROC)were used to evaluate the separate and combined diagnostic performance of serum miRNAs and serum exosomal miRNAs for the early diagnosis of NSCLC patients.2.Mimics and inhibitor for serum exosomal miRNAs associated with the early diagnosis of NSCLC were constructed and transfected into NSCLC cells,respectively.The expression of miRNAs in NSCLC cells was enchanced by mimics,while the expression of miRNAs in NSCLC cells was silenced by inhibitor.The CCK-8,wound healing assay,transwell assay and flow cytometry were used to detect the effect of miRNA overexpression/knockdown on viability,migration,invasion and apoptosis of NSCLC cells.Results1.The levels of serum miR-21-5p,miR-141-3p,miR-222-3p,and miR-486-5p in early NSCLC group were significantly higher than that in LBL group and HC group(P<0.01).The AUC of serum miR-486-5p was 0.697,and the other 3 serum miRNAs were >0.700,of which miR-222-3p had the highest AUC of 0.779,with sensitivity of 60.0% and specificity of 91.7%.The AUC of combination of 4 serum miRNAs was up to 0.871.2.The levels of serum exosomal miR-146a-5p and miR-486-5p in early NSCLC group were significantly higher than that in LBL group and HC group(P<0.01).The AUCs of serum exosomal miR-146a-5p and miR-486-5p were 0.813 and 0.886,respectively,and the AUC of combination of 2 serum exosomal miRNAs was 0.898.3.Among the 3 combinations of 4 serum miRNAs with serum exosomal miR-146a-5p and/or miR-486-5p,the combination of 4 serum miRNAs with serum exosomal miR-146a-5p and miR-486-5p had the highest diagnostic performance for early NSCLC,with an AUC of 0.960,sensitivity of 85.4% and specificity of 92.5%.4.Compared with the Blank/negative control groups,the cell viability,migration,and invasion abilities of A549 cells with overexpression of miR-146a-5p were significantly reduced,and the cell apoptosis rates was significantly increased.On the other hand,the cell viability,migration,and invasion abilities of A549 cells with knockdown of miR-146a-5p were significantly enchanced,and the cell apoptosis rates was significantly increased.5.Compared with the Blank/negative control groups,the cell viability,migration,and invasion abilities of A549 cells with overexpression of miR-486-5p were significantly reduced.On the other hand,the cell viability,migration,and invasion abilities of A549 cells with knockdown of miR-486-5p were significantly enchanced.The apoptosis rates of NSCLC cells with both overexpression and knockdown miR-486-5p were not significantly different from that of the Blank/ negative control groups.Conclusions1.Serum miR-21-5p,miR-141-3p,miR-222-3p,and miR-486-5p and serum exosomal miR-146a-5p and miR-486-5p have good potentials for the early diagnosis of NSCLC.Other than serum miRNAs,serum exosomal miRNA might be preferable biomarkers for the patients with NSCLC at early stage,and combination of serum miRNAs with serum exosomal miRNAs contribute to the further improvement of early diagnosis for NSCLC.2.MiR-146a-5p and miR-486-5p might be potential tumor suppressors for NSCLC.Compared with miR-486-5p,miR-146a-5p not only significantly inhibits the viability,migration and invasion of NSCLC cells,but also significantly induces the apoptosis of NSCLC cells,which has shown more significant inhibitory effect on malignant biological behavior of NSCLC cells.