| Research purposes In this study,we found differentially expressed mRNAs by Next Generation Sequencing of exosomal RNA extracted from the urine of bladder cancer patients and healthy individuals.Subsequently,the differential expression verification and diagnostic efficacy assessment of the target mRNAs were performed with a certain number of samples to obtain bladder cancer biomarkers with clinical diagnostic value.Research methodUrine samples were collected from 150 newly diagnosed patients with bladder cancer who had not undergone any drug or surgical treatment and 133 healthy subjects with no abnormal physical examination results admitted to the Urology Surgery and Physical Examination Center of Sun Yat-sen Memorial Hospital and Shanghai 10 th People’s Hospital from April 28,2021 to January 12,2023.Urinary exosomes were enriched by10% PEG6000 and the validity of exosome extraction was verified by western blotting,Nanoparticle Tracking Analysis and Transmission Electron Microscope.This study include discovery stage,preliminary verification stage,training stage and verification stage.The discovery stage revealed differentially expressed genes in the two groups by sequencing urinary exosomal RNA from 15 bladder cancer patients and 5 healthy individuals.In the preliminary validation stage,the differentially expressed mRNA in urine exosomes of 24 bladder cancer patients and 17 healthy people was verified by q RTPCR,and the target genes were initially identified.In the training stage,the differential expression of urinary exosome target genes in 57 patients with bladder cancer and 53 healthy subjects was verified by q RT-PCR.Then,the independent and combined diagnostic efficacy of the target genes for bladder cancer was evaluated by logistic regression analysis and ROC curve drawing,and the diagnostic model was established.In the validation stage,the efficacy of the diagnostic model established in the training stage was verified based on the sample cohort of 61 patients with bladder cancer and 51 healthy people.Research resultThe results of western blotting,Nanoparticle Tracking Analysis and Transmission Electron Microscope all confirmed the successful extraction of urine exosomes.In the discovery stage,five mRNAs differentially expressed in bladder cancer patients and healthy people,including KRT17,GPRC5A,DUSP5,STMN1 and GALNT1,were selected through analysis of sequencing data.In the preliminary verification stage,the expression levels of differentially expressed mRNAs were verified by q RT-PCR.The expression levels of the above 5 genes in bladder cancer patients were higher than those in healthy people,and the difference was statistically significant(P<0.05).The differential expression of these five genes was also verified in the training stage,where STMN1 and GALNT1 were not differentially expressed between bladder cancer patients and healthy subjects(P>0.05).Binary univariate and multivariate logistic regression analysis and ROC curves were performed for KRT17,GPRC5A and DUSP5,and the results showed that the efficacy of the combined diagnosis of KRT17+GPRC5A(AUC =0.894)was not only better than that of KRT17(AUC = 0.893),GPRC5A(AUC = 0.764)and DUSP5(AUC = 0.718),but also the combined diagnostic efficacy of KRT17+DUSP5(AUC = 0.889),GPRC5A+DUSP5(AUC = 0.753),and KRT17+GPRC5A+DUSP5(AUC = 0.881).Therefore,the diagnostic model logit(BCA)of KRT17+GPRC5A = 0.0057010×KRT17-0.0011460×GPRC5A-1.72626 was established,and the model was validated by a certain number of cohort data in the validation phase(AUC = 0.850).The model has good diagnostic efficacy with good discrimination between bladder cancer patients and healthy individuals.ConclusionThe 10% PEG6000 used in this study successfully enriched urine exosomes.KRT17,GPRC5A and DUSP5 have good discrimination between bladder cancer patients and healthy people,and have good diagnostic efficacy for bladder cancer.The combined diagnosis efficiency of KRT17 and GPRC5A is better than that of single target alone,the combination of other targets in pairs,and the combined diagnosis of three targets,which has certain clinical application value. |
