Identification Of Serum-Derived Exosomal MiRNAs As Potential Biomarkers For Early Diagnosis And Efficacy Evaluation Of Tranditional Chinese Medicine Of Non–Small Cell Lung Cancer

Non-small cell lung cancer(NSCLC)as the most common type accounts for about 80%lung cancer with a low five-year survival rate because of untimely and ineffective diagnosis and delayed treatment.Specific biomarkers especially from the serum for early diagnosis of NSCLC and adenocarcinoma(AC)/squamous cell carcinoma(SCC)discrimination are still lacking.At present,the clinical application,such as chest X-ray,low-dose spiral pulmonary CT,bronchomucosal biopsy or lung biopsy is mainly used for the examination of lung cancer.However,due to the poor sensitivity and specificity,increased medical radiation risk,high risk of invasive examination and other defects,the application is limited to a certain extent.Furthermore,miRNAs have been widely studied in lung cancer early diagnosis although the stability and repeatability of miRNAs in clinical application need to be further validated and explored.Exosome and exosomal contents are recognized as biomarker for noninvasive early diagnostic,prognosis,monitoring response to treatment,and potentially to personalize therapy in cancer.Almost all types of cells can secrete exosomes,including reticulocytes,dendrocytes,reticulocytes,and endothelial and epithelial cells.Exosomes can also be isolated from different biological fluids,such as blood,bronchoalveolar lavage,urine,nasal secretions,pleura effusions,and ascites.Exosomal miRNAs are enriched in the circulatory system and are remarkably stable compared to extracellular miRNAs because of exosomes protection it from RNase degradation.At present,surgical resection is the main treatment method for NSCLC,but because 75%of lung cancer is diagnosed at advanced stage or already has distant metastasis,chemotherapy can only be used for treatment.However,the side effects of chemotherapy drugs are large and the drug resistance of chemotherapy drugs is high.Therefore,the treatment of lung cancer with traditional Chinese medicine has received extensive attention.Ginsenoside Rg3,sodium norcantharidate and p-elemene are widely used monomers of traditional Chinese medicine for the treatment of lung cancer.At present,the main method for monitoring response to treatment of lung cancer is still lung CT.However,due to the different mechanism of drug for lung cancer,tumor volume of some kind lung cancer will increase after drug treatment,which may lead clinicians to make wrong judgments on the development of lung cancer.As a circulating biomarker,miRNAs have become a hot topic of research in recent years.Studies have found that miRNAs can be used as therapeutic evaluation biomarkers,while miRNAs in exosomes have a higher concentration and better stability.Objectives:1.To screen the serum exosomal miRNAs as biomarkers for the early diagnosis of NSCLC and the differentiation of AC and SCC.2.To quantitatively analyze the exosome miRNAs(exosome miRNAs which be screened in part one)expression which derived from NSCLC cells before and after the therapy by Chinese medicine monomers,and screen exosome miRNAs that can be used to biomarkers for assess response to treatment.Methods:1.Patients and samples:three groups(1)Profile screening:miRNA microarray was used to identify 12 NSCLC patients(6 of AC and 6 of SCC)and 6 healthy volunteers.Exosomal miRNAs expression profiles were examined using Affymetrix miRNA 4.0 microarray,according to the manufacturer’s protocol.(2)Training group:To validate the exosomal miRNAs profile and evaluate miRNA expression by RT-qPCR in serum exosomes,another 23 NSCLC patients(13 AC and 10 SCC)and 20 healthy individuals with similar status to the training group were studied.(3)Test group:the accuracy of target miRNAs as early NSCLC diagnostic biomarkers was measured by RT-qPCR in an independent cohort including 80 NSCLC patients(48 of AC and 32 of SCC)and 40 normal persons.2.To isolate exosomes from serum with high-speed centrifugation,the characteristic of exosomes derived from serum samples were determined by electron microscopy,exosomes were also characterized by the exosome-specific expression of CD 9 and CD 63 by western blot.3.Serum exosomal miRNAs was isolated using Ambion mirVanaTM miRNA Isolation Kit,according to the protocol.Exosomal miRNAs expression profiles were examined using Affymetrix miRNA 4.0 microarray,evaluate miRNA expression by RT-qPCR.And at the same time,we demonstrated the expression of target miRNAs in serum exosomes and circulating miRNAs simultaneously(n=59).4.To isolate exosomes from serum with high-speed centrifugation,the characteristic of exosomes derived from serum samples were determined by electron microscopy,exosomes were also characterized by the exosome-specific expression of proteins by western blot.5.The optimal concentration and time of effective of ginsenoside Rg3,sodium cantharidate and p-elemene on AC cells(A549,H1975)and SCC cells(H226,H1915)were screened by CCK-8 method.6.Rt-qPCR analysis was used to assess the expression of miRNAs(miRNAs screened for early diagnosis of non-small cell lung cancer)in exosomes before and after the dealing with Chinese medicine monomers.Results:1.The miRNAs which can be discriminated the healthy groups from AC and SCC groups were identified using Affymetrix miRNA 4.0 microarray by hierarchical clustering analyses.61 upregulated and 26 downregulated miRNAs were defined to be further analysis depending on differentially expressed analysis with>3 folds change for NSCLC vs Control.And 13 upregulated and 103 downregulated miRNAs expressed differentially according to the same chosen criteria in comparison to AC and SCC.As a result of hierarchical clustering analyses,a distinct miRNA signature,6 upregulated(miR-486-3p,miR-455-3p,miR-3201,miR-4516,miR-3613-5p,miR-8084)and 9 downregulated miRNAs(miR-342-3p,miR-199a-3p,miR-22a-3p,miR-140-3p,miR-18a-3p,miR-27a-3p,miR-221-3p,miR-6126,miR-6715a-3p,miR-139-5p)in NSCLC patients,4 upregulated(miR-150-3p,miR-500a-3p,miR-143-3p)and 3 downregulated miRNAs in AC compared with SCC patients,respectively,were selected for further validation.2.The qPCR results showed that top 7 miRNAs including miR-4516,miR-6715a-3p,miR-6126,miR-15b-5p,miR-150-3p,miR199a-3p,and miR195-3p were significantly consistent expressed with training set.Four of these miRNAs(miR-4516,miR-6715a-3p,miR-6176,miR-199a-3p)were dramatically differently expressed between NSCLC and CRT,miR-15b-5p,miR-150-3p and miR-195-3p were specific to distinguish AC and SCC patients.3.Our ROC analysis results showed that the area under the curve(AUC)of miR-4516,miR-6715a-3p,miR-6126,miR-15b-5p,miR-150-3p,miR199a-3p,and miR195-3p in the test set.The results showed that AUC of miR-4516 was 0.956(95%confidence interval(CI)=0.9295 to 0.9893;sensitivity=87.5%,specificity=90.0%),the AUC of miR-6715a-3p was 0.932(95%CI=0.8825 to 0.9814;sensitivity=87.8%,specificity=87.65%),respectively.The three miRNAs for NSCLC early diagnosis exhibited an obviously reliable higher AUC value.These 2 miRNAs could serve as primary diagnostic biomarkers for NSCLC.Moreover,we found that miR-15b-5p and miR-150-3p for the diagnosis exhibited an AUC value of 0.888-0.8964 in distinguishing AC from SCC patients with a sensitivity of 78.8-81.8%and a specificity of 83.7-85.7%.4.Negative correlations between the miRNA expression levels in serum exosomes and circulating miRNAs from the same individuals were observed in miR-6715a-3p.No correlations between the serum exosomal miRNAs and circulating miRNAs were observed in miR4516,miR-15b-5p and miR-150-3p from the same individuals.5.All three monomers of traditional Chinese medicine have certain inhibitory effects on NSCLC cells.The optimal concentration and time of ginsenoside Rg3 on A549,H1975 and H226 are respectively 60 μg/mL and 48 hours,while the optimal concentration and time for H1915 are respectively 60 μg/mL and 72 hours.The optimal concentration and time of β-elemene on the four cell lines were 200 μg/mL and 48 hours,respectively.The optimal concentration and time of sodium norcantharidate on the four cell lines were 4 μg/mL and 72 hours respectively.6.After the addition of ginsenoside Rg3 to NSCLC cell lines(A549,H226,H1915),the relative expression of miR-6715a-3p decreased compared with before the addition,with statistically differences.The relative expression of miR-4516 in NSCLC cell lines after the addition of β-elemene,was up-regulated compared with before the addition β-elemene,with statistically significant difference.After the addition of Sodium Norcantharidate on the NSCLC cell line,miR-4516 showed upregulated expression compared with before the addition of drugs,with statistical difference.Conclusions:1.The three miRNAs including miR-4516 and miR-6715a-3p for NSCLC early diagnosis exhibited an obviously reliable higher AUC value.These 3 miRNAs could serve as primary diagnostic biomarkers for NSCLC.2.The miR-15b-5p and miR-150-3p for the diagnosis exhibited a higher AUC value in distinguishing AC from SCC patients.This result demonstrated the two miRNAs could be differential diagnosis biomarker for AC or SCC.3.MiR-6715a-3p derived from exosomes may be a biomarker for the efficacy evaluation for ginsenoside Rg3 in NSCLC(A549,H226,H1915)treatment.4.MiR-4516 derived from exosomes may be a biomarker for the efficacy evaluation for p-elemene and sodium norcantharidate in NSCLC treatment.