Urine Exosome MRNA For Early Diagnosis Of Prostate Cancer

Objective: Through the RNA sequencing analysis of urine exosomes in patients with prostate puncture,urine exosome mRNA with significant differential expression was selected,and a comprehensive diagnostic model based on urine exosome mRNA was constructed to explore its value in the early diagnosis of prostate cancer(PCa),To reduce unnecessary prostate puncture,and provide a basis for the development of corresponding clinical testing kits.Methods: Use ultracentrifugation to extract exosomes in urine samples,and use next-generation sequencing technology to sequence and analyze the urine exosome RNA to obtain mRNA with significantly differential expression;According to the expression level,the top 10 mRNAs with the most significant differences related to prostate cancer were selected as candidate molecular markers,and then the distribution characteristics of these 10 mRNAs in urine exosomes were further explored;Design probes and primers specifically for the exon of the target gene,and obtain a high-abundance and single-band exosome RNA fragment by comparing the expression levels of RT-PCR products;A large number of clinical urine exosome samples(43 cases of PCa and 92 cases of benign prostatic hyperplasia)were further verified,and the relative expression levels of candidate mRNAs in the two independent study populations were detected by q RT-PCR technology;Use univariate logistic regression analysis to predict the independent risk factors of PCa;multivariate logistic regression analysis to construct a molecular diagnostic model of PCa;The receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of relevant indicators and models.Results: Successful separation of stable exosomes by ultracentrifugation;We explored the distribution of ten candidate molecules in exosome RNA,through the analysis of target gene fragments,multiple detection primers were designed for each mRNA,and RT-PCR technology was used to find the PCR products with the highest expression abundance and single band in each target gene,and specifically designed the detection Probe,after the probe verification,8 exosome genes were finally obtained,namely WWP1,RBM10,ZFY,NRSN2,MARK2,PASK,HIST2H2 BF,RAB5B;further expanded clinical urine samples to verify the above 8 target genes,q RT-PCR results It showed that the relative expression of 8 target genes in urine exosomes of PCa group was higher than that of benign prostatic hyperplasia group,the difference was statistically significant(all P<0.001);The results of univariate logistic regression analysis indicated that these 8 target genes are all independent risk factors for PCa.ROC curves show that they have good diagnostic value for PCa,Their AUCs are WWP1(0.906),RBM10(0.817),and ZFY(0.799).),NRSN2(0.856),MARK2(0.801),PASK(0.831),HIST2H2BF(0.835),RAB5B(0.880);ROC results of a comprehensive model of urine exosome mRNA constructed based on multivariate logistic regression analysis showed that the comprehensive model had an AUC of 0.921,a sensitivity of 76.7%,and a specificity of 96.7%,the positive predictive value and negative predictive value were 91.4% and 89%respectively,and urinary exosome RNA has extremely high value in the early clinical diagnosis of PCa.Conclusion: In this study,based on the analysis of urine exosome sequencing,we successfully screened eight significantly exaggerated PCa-related exosome genes,including WWP1,RBM10,ZFY,NRSN2,MARK2,PASK,HIST2H2 BF,RAB5B,further confirming that these eight target genes are independent predictors of PCa and can be used as potential markers for early diagnosis of PCa.A comprehensive diagnostic model of urine exosome mRNA based on logistic regression can improve the early diagnosis efficiency of PCa.