Preliminary Study Of Plasma Exosomal MiRNA In The Diagnosis And Evaluation Of Gastric Cancer

Objective:This study aimed to analyze the characteristics of exosomal microRNA(miRNA)expression profiles in plasma from patients with gastric cancer(GC).Exosomal miRNAs and exosomal miRNA modules related to clinical pathological characteristics were identified and analyzed to find potential biomarkers for diagnosis and evaluation of GC.Methods:24 patients with GC admitted to the First Affiliated Hospital of Soochow University from September 2018 to December 2018 were collected.Six healthy volunteers that underwent physical examinations at the physical examination center were collected as controls.ExoEasy Maxi Kit was used to extract plasma exosomes,and transmission electron microscope(TEM)imaging method was used to identify exosomes.After small RNA high-throughput sequencing,miRNA sequence identification used bowtie software and Mirdeep2 software.The expression of miRNA was calculated using TPM(transcript per million).Exosomal miRNA differential gene screening,principal component analysis(PCA),weighted gene co-expression analysis(WGCNA),GO and KEGG functional enrichment analysis,and miRNA expression verification of gastric cancer tissue in TCGA database were performed based on R software.Target genes of key miRNAs were predicted by mapping the miRNA target gene prediction databases(miRDB,miRTarBase and TargetScan).The diagnostic efficacy of differential exosomal miRNAs was evaluated by calculating the receiver working curve(ROC).Cytoscape was used to assist data visualization.Results:The TEM imaging identified that the plasma exosomes extracted from this study were about 100 nm in diameter.Among the sequences obtained by high-throughput sequencing,a total of 1853 plasma exosomal miRNAs were annotated,of which 1114 were known miRNAs and 739 were novel miRNAs.The PCA model established by 31 differentially expressed plasma exosomal miRNAs screened from GC patients could significantly distinguish the GC patients from the healthy controls.The expression of plasma exosomal hsa-miR-200a-3p was up-regulated in GC patients of stage III+IV,which was significantly higher than those in the healthy controls(FC=32.21,P=0.02)and GC patients of stage Ⅰ+Ⅱ(FC=3.96,P<0.01).The expressions of plasma exosomal hsa-miR-320d and hsa-miR-3613-5p were significantly up-regulated in the GC patients with distant metastasis,which were higher than those in the healthy controls(FC=9.78,3.59,P=0.03,0.02)and GC patients without distant metastasis(FC=5.75,3.65,P<0.01,P=0.049).The expression of plasma exosomal hsa-miR-451a was significantly down-regulated in the GC patients with lymph node metastasis,which was lower than those in the healthy controls(FC=0.47,P=0.01)and the GC patients without lymph node metastasis(FC=0.43,P=0.01).Seven plasma exosomal miRNAs including hsa-miR-3661 and hsa-miR-1249-3p showed great diagnostic value of the diagnosis of GC(AUC>0.8).Plasma exosomal hsa-miR-451a showed diagnostic value of the diagnosis of GC with lymph node metastasis(AUC=0.780),while plasma exosomal hsa-miR-200a-3p and hsa-miR-3613-5p showed great diagnostic value of the diagnosis of GC with distant metastasis(AUC=0.850,0.858).WGCNA revealed ME blue and ME yellow,two plasma exosomal miRNA modules,were positively associated with distant metastasis.Within the modules,hsa-miR-3613-5p and its co-expressed miR-371-373 family members,hsa-miR-200a-5p and its co-expressed miR-200 family members were all significantly positively related to distant metastasis.Functional enrichment analysis showed that the target genes of plasma exosomal hsa-miR-3613-5p and its co-expressed plasma exosomal miRNAs were highly enriched in SMAD binding,PI3K-Akt signaling pathway,MAPK signaling pathway,RAS signaling pathway.While the target genes of plasma exosomal hsa-miR-200a-3p and its co-expressed plasma exosomal miRNAs were highly enriched in cell junction assembly and organization,regulation of cell morphogenesis,regulation of epithelial cell differentiation,cell-substrate adhesion junctions.Conclusion:This study identified several key plasma exosomal miRNAs which were closely related to the clinical pathological characteristics of GC.They might have potential clinical applications for the diagnosis and evaluation of GC,and might participate in the occurrence and development of GC.