Exosomal MiR-1246 In Serum As A Potential Biomarker For Early Diagnosis Of Gastric Cancer

Background: Gastric cancer(GC)is one of the high incidence of malignant tumors in the world.Currently,gastroscopy is the standard method to diagnose GC,with high safety,but it is difficult to accept as an invasive examination for some people.Some tumor markers have been widely used in clinic,but their sensitivity and specificity need to be improved.Non-invasive low-cost biomarkers are needed for early detection of GC.Micro RNAs(miRNAs)are a class of endogenous non-coding RNAs that regulate gene expression by degrading target m RNAs or blocking their translation after transcription.Exosomes are a group of vesicles composed of lipid bilayer membranes that contain a series of components from mother cells,such as miRNAs.Studies have demonstrated the value of circulating exosome miRNAs as biomarkers in the diagnosis and prediction of prognosis in patients with tumors.Objective:1.Mi R-1246 was screened as an early detection of gastric cancer,and its possibility and value as an early detection of gastric cancer was further verified.2.The production and metastasis pathways of miR-1246 were analyzed to explore its relationship with gastric cancer cells and its mechanism of action.3.Potential target proteins,pathway enrichment analysis and protein interactions of miR-1246 were analyzed.Methods: From October 2017 to September 2018,165 fresh blood samples and 32 pairs of tissue samples were collected.Among the blood samples,85 cases were gastric cancer,50 cases were healthy control group(HC),and 30 cases were benign gastric disease.The tissue samples included gastric cancer tissues and adjacent normal tissues(ANT).A comprehensive database of gene expression and blood samples were analyzed to screen miRNA with high expression in gastric cancer.Exosomes extracted from serum were verified by using transmission electron microscopy(TEM)and western blot.The expressions of miRNAs were tested through q RT-PCR.The correlation between miR-1246 expression and clinical data(TNM stage,metastasis and tumor differentiation)of 85 gastric cancer patients was analyzed.The expression of miR-1246 in gastric cancer and paracancer tissues was analyzed.Gastric cancer cell lines were cultured,exosomes at multiple time points were collected,and observed by TEM and western blot.The mechanism of transport of miR-1246 to exosomes was detected by RNA drop-down assay.Bioinformatics analysis of miR-1246 was carried out by DAVID,Cytoscape and other software to search for potential targets and signaling pathways involved in regulation.Candidate miRNA was identified after integrated analysis of two Gene Expression Omnibus(GEO)datasets and clinical serum samples.Exosomes extracted from serum were verified by using transmission electron microscopy(TEM)and western blot.The expressions of miRNAs were tested through q RT-PCR.Receiver operating characteristic curve(ROC)analysis was used to explore the diagnostic utility of miRNAs.Exosomes extracted from gastric cancer cell were verified by using transmission electron microscopy(TEM)and western blot,RNA pull-down assay was used to find themechanism by which miRNAs were transported into candidate exosomes.Bioinformatics analysis of candidate miRNA was conducted by using softwares such as DAVID and Cytoscape,to find potential targets and signal pathways involved in regulation.Results: By comprehensive analysis of the two GEO data sets,we found that 6miRNAs were continuously up-regulated in the circulating blood of patients with gastric cancer,and analysis of the data showed that miR-1246 was the one with the largest fold change.Compared with other miRNAs,the difference of serum miR-1246 in GC patients and HC was statistically significant(P < 0.05).The results of qrt-PCR showed that the expression of serum exosome miR-1246 was significantly up-regulated in GC,and the difference between the two groups was statistically significant(P < 0.05).In addition,the expression of serum exosome miR-1246 was significantly down-regulated after tumor resection,which was statistically significant compared with the preoperative group.Compared with ANTs,the expression of miR-1246 in GC tissues was significantly down-regulated and the difference was statistically significant.The expression of miR-1246 in serum exosomes can distinguish GC patients from HC.ROC analysis demonstrated that circulating exosomal miR-1246 expressions could differentiate GC patients in TNM stage I from HC with an AUC value of 0.843(95% CI:0.75-0.93,P<0.001).The sensitivity and specificity were 85.71% and 74%,respectively.The cut-off value was 1.43.Compared to patients with BD,circulating exosomal miR-1246 levels were also significantly higher in GC patients with TNM stage I.ROC analysis demonstrated that circulating exosomal miR-1246 expressions could differentiate GC patients in TNM stage I from BD patients with an AUC value of 0.811(95% CI:0.71-0.93,P<0.001).The sensitivity and specificity were 78.57% and 80%,respectively.The cut-off value was 1.53.In addition,circulating exosomes gradually increased in patients with stage III-IV,and the level of miR-1246 was significantly higher than that of patients with stage I-II.The 85 patients with gastric cancer were divided into high and low groups according to the median expression of external miR-1246.Chi-square test showed that the highly expressed external miR-1246 was significantly correlated with distant metastasis(P = 0.035).In vitro experiments showed that the expression of miR-1246 in the medium increased with time and the number of cells.After the treatment of AGS cells with GW4869,the expression of miR-1246 in the medium was significantly down-regulated and transferred to exosomes by binding to ELAVL1.GO enrichment analysis showed that the target genes of miR-1246 were significantly enriched in the positive regulation of cell proliferation,positive regulation of transcription,positive regulation of gene expression,and negative regulation of palatal development and apoptosis.CC analysis showed that most of the targets were located in the cell membrane.According to MF analysis results,the target of miR-1246 is mainly related to protein binding,including binding of transformed growth factor and binding of growth factor.Enrichment analysis of KEGG signaling pathway revealed that miR-1246 was involved in a variety of signaling pathways,including Hippo signaling pathway,Fox O signaling pathway,PI3K-Akt signaling pathway,and Gn RH signaling pathway.Conclusion: 1.Exosome miR-1246 in peripheral blood can be used as a potential biomarker marker for early diagnosis of gastric cancer.2.Elevated miR-1246 in the serum was encapsulated in exosomes with the help of ELAVL1.3.The target of miR-1246 is mainly located on the cell membrane and is involved in the regulation of several signaling pathways.