The Diagnostic Performance Of Urinary Exosomal MiRNA And The Mechanism Of MiR-93-5p In Bladder Cancer

ObjectiveTo selecte the urinary exosomal miRNA as novel and non-invasive biomarkers for bladder cancer based on high-throughput sequencing technique;To explore the mechanism of miR-93-5p in bladder cancer cells.Methods1.The isolation,purification and identification of urinary exosomesThe urine from baldder cancer patients and healthy control were collected and urinary exosomes were obtained by ultracentrifugation.Exosomes were identified by transmission electron microscopy,NTA and western blot.2.Select urinary exosomal miRNA as biomarker for diagnosis and staging of bladder cancer by high-throughput sequecing.Combining high-throughput sequencing and data from TCGA,miRNA that were both significantly differentially expressed in urinary exosomes(bladder cancer vs healthy control,MIBC vs NMIBC)and tissue(bladder cancer tissue vs adajacent nomal tissue)were selected as candidate biomarkers for non-invasive diagnosis.The candidate miRNA were further validated in the clinical cohort by RT-qPCR technology.ROC curve was constructed to evaluate the diagnostic performance of candidate miRNA in bladder cancer.3.Prediction of miRNA target genes and bioinformatics analysisThe target genes of the candidate miRNA were predicted by mirwalk database,analyzed by bioinformatics analysis,such as GO enrichment analysis,KEGG pathway analysis,PPI network construction,survival analysis,to predict the possible mechanism of miRNA and target genes in the occurrence and development of bladder cancer,and to selecte the miRNA and hub genes related to the development and prognosis of bladder cancer.4.The mechanism of miR-93-5p in bladder cancerBased on bioinformatics analysis,it is found that BTG2 may be the hub target gene of miR-93-5p in the occurrence and progression of bladder cancer,and survival analysis also shown that the expression of BTG2 is related to the prognosis of patients with bladder cancer.The effect of miR-93-5p on bladder cancer cells and the targeting relationship with BTG2 were verified by CCK-8 test,transwell test,western blot,RT-qPCR and so on.Result1.Based on urinary exosomal miRNA sequencing and data analysis from TCGA,we selected miR-93-5p,miR-516a-5p and miR-940 as candidate biomarkers.The results of RT-qPCR showed that miR-93-5p and miR-516a-5p were highly expressed in urinary exosomes from bladder cancer patients compared with healthy control(p<0.01),but there was no significant difference in miR-940 expression(p>0.05).The expression of urinary exosomal miR-93-5p in patients with MIBC was higher than that in patients with NMIBC(p<0.01),but there was no significant difference in miR-516a-5p between patients with MIBC and NMIBC(p>0.05).2.The results of ROC curve showed the potential of urinary exosomal miR-93-5p and miR-516a-5p to distinguish bladder cancer patients from healthy people,and the areas under the curve were 0.832 and 0.798 respectively.Mir-93-5p also show the potential to distinguish MIBC from NMIBC,and the area under the curve was 0.755.3.The results of bioinformatics analysis showed that the target genes of miR-93-5p may play a role in the occurrence and development of bladder cancer through FoxO signal pathway,MAPK pathway,PI3K-Akt pathway and EGFR tyrosine kinase inhibitor pathway and so on,while the target genes of miR-516a-5p play a role through calcium signal pathway,MAPK pathway,JAK-STAT pathway and so on.BTG2 is the hub target gene of miR-93-5p,and its expression level is related to the prognosis of patients.4.The expression of miR-93-5p can specifically inhibit BTG2,and promote the proliferation,invasion and migration of bladder cancer cells.ConclusionWe identified miR-93-5p and miR-516a-5p in urinary exosomes as potential liquid biospy indicators for bladder cancer patients,and miR-93-5p promotes the proliferation,invasion and migration of bladder cancer through targeting BTG2.