A Preliminary Study Of Bronchoalveolar Lavage Fluid Exosomal MiRNAs In The Diagnosis Of Non-small Cell Lung Cancer

Objective1.To prove that(bronchoalveolar lavage fluid,BALF)exocrine bodies in bronchoalveolar lavage fluid can be obtained by ultracentrifugation.2.To obtain exosome miRNA with different expression among groups.3.To complete the GO enrichment analysis and KECG pathway enrichment analysis of differential miRNA target genes,and to complete the preliminary study on the prediction results of differential miRNA target genes.Method1.The bronchoalveolar lavage fluid of inpatients was collected and the exocrine was extracted by ultracentrifugation.According to inclusion and exclusion criteria,the samples were divided into lung adenocarcinoma group,lung squamous cell carcinoma group and normal control group.2.Three methods were used to identify the exosome,including morphological were observed by Transmission Electron Microscope(TEM),particle size determination were detected by Nanoparticle Tracking Analysis(NTA)and exosome specific protein were identified by Western blotting(WB).3.We should complete the extraction and quality control of exocrine total RNA samples,establish a sequencing library and sequence exosomal miRNA,and then evaluate the sequencing results.4.Bioinformatics software was used to analyze the miRNA differences between groups,and its molecular biological function.Results1.The exosome of BALF were successfully extracted by ultracentrifugation,and the typical cup-shaped bilayer membrane structure was observed under TEM.The particle size of more than 60% was between 30-150 nm.WB analysis showed that each group of samples contained exosome marker proteins(CD9,CD63 and CD81).It was observed that the concentration of exocrine in the control group was lower than that in the lung cancer group.2.Three important quality control results meets the sample requirements,including the quality control of exosomal total RNA samples,quality control of sequencing library and the quality control of library preparation.3.The differentially expressed miRNA was screened out.Four different kinds miRNA were significantly down-regulated in lung adenocarcinoma,including hsa-let-7b-5p,hsa-miR-92b-5p,hsa-miR-200c-3p and hsa-miR-27b-3p;six different kinds miRNA were significantly up-regulated in lung squamous cell carcinoma group,including hsamiR-200c-3p,hsa-miR-143-3p,hsa-miR-532-5p,hsa-miR-27b-3p,hsa-miR-891a-5p and hsa-let-7g-5p.4.Three levels of functional annotations were obtained by GO enrichment analysis of differential miRNA.The differential miRNA down-regulation in adenocarcinoma involves Biological Process(BP)including neuron projection guidance,regulation of cell morphogenesis involved in differentiation and regulation of axonogenesis.Cellular Component(CC)includes postsynaptic density,asymmetric synapse and glutamatergic synapse,Molecular Function(MF)includes protein serine/threonine kinase activity.The differential miRNA up-regulated in squamous cell carcinoma involves the following functions: positive regulation of cell projection organization,stress-activated MAPK cascade,stress-activated protein kinase signaling cascade and so on.CC includes cell leading edge,neuron to neuron synapse and collagen trimers,and MF includes small small GTPase binding and protein serine/threonine kinase activity.5.The metabolic pathway information of different miRNA target genes was obtained by KEGG database.The down-regulated miRNA in lung adenocarcinoma group is mainly involved in MAPK signal pathway and cancer-related pathway(pathway in cancer),while the up-regulation of miRNA in lung squamous cell carcinoma is mainly related to glucose metabolism pathway and Herpes Simplex Virus(HSV)infection signal pathway.6.Targetscan is used to predict the relationship between different miRNA target genes.NEK8,NOTCH2 NL,ZNF28 and other genes are the direction of further research.Conclusion1.Exosomes were extracted from BALF by ultrafentrifugation and identified by TEM,NTA and WB.2.The quality evaluation of miRNA results of high-throughput sequencing of samples meets the requirements of follow-up experiments.The results of volcano map and cluster analysis showed the similarities and differences of miRNA among samples.3.Ten significant differentially expressed miRNAs are selected by significant differences between groups,GO function enrichment,KEGG pathway enrichment analysis and target gene prediction and are performed based on these expressed miRNAs.