Exosome-encapsulated MiRNAs In Urine As A Non-invasive Biomarker For Prostate Cancer Diagnosis

Prostate cancer is one of the most common malignant tumors in male urinary system,and it is also the second largest killer of male cancer.Although the conventional biopsy is the gold standard for the diagnosis of tumor,it has some limitation in application because it is an invasive procedure.Secondly,PSA is the most commonly used biomarker for prostate cancer screening in spite of the problem related to its low specificity.On the other hand,PSA screening can lead to over diagnosis and over treatment of indolent prostate cancer Therefore,there is an urgent need for new and non-invasive biomarker for early diagnosis of prostate cancer with high sensitivity and specificity.Exosomes are membrane vesicles in the range of 30–100 nm in diameter,which are secreted by various mammals.They widely exist in variety of body fluids including blood,urine,saliva,semen and contain specific proteins,m RNAs and mi RNAs.A large number of studies have confirmed that mi RNA can transmit information between various tumor cells by exosome,and aberrant expression of mi RNAs can reflect the physiological and pathological state of the body.Recent studies have shown that mi RNAs derived from urine exosome reflect their cellular origin,therefore,mi RNAs enclosed in the urine exosome may be a new non-invasive molecular marker for the diagnosis of many cancers,particularly the prostate cancer.Based on this,in order to explore the diagnostic value of urinary exosomal mi RNA for prostate cancer,we conducted the following two parts:Part 1 The diagnostic value of urine exosomal mi RNAs in prostate cancerObjective: To investigate the clinical value of urinary exosomal mi RNA in prostate cancer.Methods: We screened the differentially expressed urine exosomal mi RNAs by second-generation deep sequencing using pooled urine samples from 6 PCa patients(3 localized prostate cancer patients and 3 metastatic prostate cancer patients)and 3 healthy controls.Significantly deregulated exosomal mi RNAs in urine samples were further validated by q RT-PCR in the cohort consisting of 47 PCa patients and 25 healthy controls.In the meanwhile,the expression of urine exosomal mi RNA was detected by q RT-PCR in 29 patients with benign prostatic hyperplasia.Results:1.A genome-wide expression profiling of exosomal mi RNAs obtained by Illumina NGS technology showed that there were 31 significantly differentially expressed exosomal mi RNAs between the prostate cancer group and the healthy control group(p<0.05),and there were 7 significantly differentially expressed exosomal mi RNAs between the localized prostate cancer group and the metastatic prostate cancer group(p<0.05).For validation,we selected 5 exosomal mi RNAs based on those mi RNAs with a mean fold-change>2 and p value<0.05 between PCa group and healthy control group,and those mi RNAs with a mean fold-change>2 and p value<0.05 between localized PCa group and metastatic PCa,group including mi R-375,mi R-16-2-3p,mi R-451 a,mi R-486-3p and mi R-486-5p.2.The q RT-PCR results demonstrated that the expression of mi R-375 was significantly downregulated in urine samples of PCa patients than that of healthy controls.On the contrary,the expression levels of exosomal mi R-451 a,mi R-486-3p and mi R-486-5p were shown to be significantly upregulated in PCa samples compared to healthy controls.However,there was no significant difference in the relative expression of miR-16-2-3p in PCa group compared to healthy controls.The expression level of mi R-375 was correlated to clinical stage and bone metastasis status of the patients with PCa,no significant relationship was detected between mi R-375 level and the patient's age,Gleason scores and serum prostate-specific antigen level(p>0.05).The expression levels of exosomal mi R-451 a,mi R-486-3p and mi R-486-5p had no relationship with age,Gleason score,clinical stage,metastasis and serum PSA level.Receiver operator characteristic analyses demonstrated that the urine exosomal mi R-375,mi R-451 a,mi R-486-3p and mi R-486-5p were able to differentiate PCa patients from healthy controls.The AUC was 0.788,0.757,0.704,0.796 respectively.3.Compared with patients with benign prostatic hyperplasia,mi R-375 was significantly lower in patients with prostate cancer(p<0.05),mi R-451 a was highly expressed in patients with prostate cancer(p<0.05).However,mi R-486-3p and mi R-486-5p had no significant difference between prostate cancer patients and patients with benign prostatic hyperplasia(p>0.05).Receiver operator characteristic analyses revealed that the urine exosomal mi R-375+mi R-451 a could differentiate PCa patients from benign prostatic hyperplasia patients,the AUC was 0.726(p<0.05).4.Compared with localized prostate cancer patients,mi R-375 was downregulated in patients with metastatic prostate cancer(p<0.05).However,mi R-451 a,mi R-486-3p and mi R-486-5p had no significant difference between localized prostate cancer patients and metastatic prostate cancer patients(p>0.05).The ROC curve showed that exosomal mi R-375 could better differentiate localized prostate cancer patients from metastatic prostate cancer patients compared with PSA,the AUC was 0.806,0.624 respectively.Conclusion: This study suggests that urinary exosomal mi R-375,mi R-451 a,mi R-486-3p and mi R-486-5p are expected to be used as non-invasive molecular diagnostic markers for prostate cancer to differentiate prostate cancer patients from healthy control,exosomal mi R-375 and mi R-451 a are expected to be used as a non-invasive molecular diagnostic marker for prostate cancer to differentiate prostate cancer patients from benign prostatic hyperplasia patients,exosomal mi R-375 is expected to be used as a non-invasive molecular diagnostic marker to differentiate metastatic prostate cancer patients from localized prostate cancer patients.Part 2 Prediction of target genes for differentially expressed mi RNAs in prostate cancer and bioinformatics analysis of predicted target genes.Objective: To predict the target genes for differentially expressed mi RNAs in prostate cancer as well as to implement related bioinformatics analysis,in order to explore the underlying mechanisms in the pathogenesis of mi R-375,mi R-451 a,mi R-486-5p and mi R-486-3p in the development and progression of prostate cancer.Methods: The target genes of differentially expressed mi RNAs in prostate cancer were predicted by bioinformatics,and the target genes were analyzed by gene ontology(GO)and signal transduction pathway enrichment.Results:1.The number of mi R-375 target genes was 381;the number of mi R-451 a target genes was 103;the number of mi R-486-3p target genes was 828;the number of mi R-486-5p target genes was 404.2.The functions of predicted target genes of mi R-375,mi R-451 a,mi R-486-3p and mi R-486-5p were enriched in kinase activity,protein kinase activity,transcription factor binding of transcription factor activity,phosphotransferase activity and chromatin binding,negative regulation of nitrogen compound metabolic process,negative regulation of cellular metabolic process,regulation of cell differentiation,regulation of transcription from RNA polymerase II promoter,Golgi apparatus part,neuron part,neuron projection,perinuclear region of cytoplasm and other biological processes and molecular functions and cellular components(p<0.01).In KEGG pathway,the predicted target genes were enriched in axon guidance,thyroid hormone signaling pathway,Notch signal pathway,Hippo signal pathway,Fox O signal pathway,non-small cell lung cancer,glioma,melanoma,prostate cancer and other signaling pathways and other human disease pathway(p<0.01).Conclusion: The result of bioinformatics showed that mi R-375,mi R-451 a,mi R-486-3p and mi R-486-5p may play an important role in the development and progression of prostate cancer by regulating PDGFRB,FOXO1,PDK1,CCND1,PDGFC,TCF7L1,IGF1 R,CREB3L2,PIK3R1,IGF1,PIK3R2,E2F2,E2F3,E2F1,MAPK3,FGFR1,RELA,BCL2.