Evaluation Of Serum Exosomal LncRNA-Based Biomarker Panel For Diagnosis And Recurrence Prediction Of Bladder Cancer

Objective:Bladder cancer(BC)is one of the most common malignancies of the urinary system.More and more studies have shown that;ncRNAs play a very important role in the occurrence and development of tumors.The purpose was to explore the clinical significance of serum exosome lncRNA in the diagnosis of bladder cancer.Long non-coding RNAs(lncRNAs)embedded in circulating exosomes may serve as diagnostic biomarkers for a variety of tumors.We aimed to develop a panel consisting of serum exosomal lncRNAs for diagnosis and recurrence prediction of BC.Methods:Exosomes are characterized by their conserved size and density as well as the presence of specific protein markers.Exosomes were isolated from serum of BC patients and healthy controls and validated by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blotting analysis.RT-qPCR was performed to analyze the expressions of 11 candidate lncRNAs using a training set(n=200)and a validation set(n=320).Receiver-operating characteristic(ROC)curves were employed to evaluate the diagnostic performance of the identified lncRNAs.Multivariate logistic regression model was used to construct an IncRNA panel.Moreover,we determined the correlation between lncRNAs and recurrence-free survival(RFS)of patients with non-muscle-invasive bladder cancer(NMIBC)by Kaplan-Meier analysis,univariate and multivariate Cox analysis.Results:TEM was used to confirm the morphology of exosomes,which should be revealed as spherical vesicles with double layer membrane structure and diameters about 100 nm.Western blotting was used to examine exosomal markers at the protein level.CD9 and TSG101 could be detected in the exosome samples but not in the EDS.NTA showed the size distribution of exosomes.Three lncRNAs(PCAT-1,UBC1 and SNHG16)were significantly up-regulated in serum exosomal samples of BC patients.In the training cohort,BCs could efficiently be distinguished from controls using PCAT-1,UBC1 and SNHG16(PCAT-1:AUC=0.753;UBC1:AUC=0.751;SNHG16:AUC=0.681).A panel was established based on these three lncRNAs.The three-IncRNA panel provided high diagnostic accuracy for BC with an the area under the ROC curve(AUC)of 0.857 and 0.826 in the training set and validation set,respectively,which was significantly higher than that of urine cytology.The corresponding AUCs of this three-lncRNA panel for Ta,T1 and T2-T4 patients were 0.760,0.827 and 0.878,respectively.Kaplan-Meier analysis showed that NMIBC patients with high UBC1 expression had significantly lower RFS(p=0.01).Univariate and multivariate Cox analysis demonstrated that UBCl was independently associated with RFS(p = 0.018).Conclusion:Three(PCAT-1,UBC1 and SNHG16)out of the 11 lncRNAs had a statistically increased expression in BC patients compared with the healthy donors.Our study established a distinctive three-lncRNA panel with considerable diagnostic value and identified lncRNA UBC1 as a potential biomarker for prediction of NMIBC recurrence.