Detection And Assessment Of Prostate Cancer MiRNAs In Urinary Extracellular Vesicles Based On Hydrostatic Filtration Dialysis

BACKGROUND&OBJECTIVEProstate cancer(PCa)is a malignant disease that endangers male health,which estimated new cases is increasing in China.At present,prostate-specific antigen(PSA)is the most commonly used PCa biomarker.However,PSA is lack of specificity which leads to over-diagnosis and over-treatment of PCa.Since such inherent limitations of PSA,intensive efforts are currently directed towards a search for novel circulating tumor biomarkers for early diagnosis and reducing unnecessary biopsy.The study of extracellular vesicles has received extensive attention.EVs are membrane vesicles released by cells for intercellular information transmission which contain nucleic acids and proteins.Cargoes in EVs can reflect their parental cells and pathological states of the organism.Thus,EVs represent an important source of potential biomarkers.EVs can be detected in body fluids such as serum/plasma,urine,cerebrospinal fluid,saliva,breast milk and so on.Urine is regarded as an ideal source of biomarkers for diseases of the kidney and urinary tract,since it can be conveniently collected in large amounts without risk to the patients.As there was no standard method for urinary extracellular vesicles(UEVs)isolation,we chose a new approach for UEVs enrichment based on hydrostatic filtration dialysis(HFD).We compared the overall performance of HFD and ultracentrifugation(UC)in UEVs isolation and miRNA detection.Subsequently,UEVs were isolated from patients with PCa,patients with benign prostate hyperplasia(BPH)and healthy individuals.Several PCa-related miRNA were validated followed by diagnostic efficiency assessment.The study can be divided into two parts as below:Part 1 Comparison of HFD and UC in UEVs IsolationOBJECTIVETo validate hydrostatic filtration dialysis(HFD)was capable to isolate urinary extracellular vesicles(UEVs)and compared the overall performance of HFD and ultracentrifugation(UC)in UEVs isolation and miRNA detection.METHODSUEVs were isolated from urine of healthy volunteers by HFD and UC respectively.Transmission electron microscopy,NanoSight,Coomassie Protein Assay Kit and Western Blot were used for estimating efficiency of UEVs isolation.Subsequently,RNA yield,RNA distribution and micro RNA(miRNA)expression levels were measured by Nanodrop 2000,Agilent 2100 and quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Differences between methods were tested using paired t-test.RESULTSHFD had a similar performance with UC in isolating UEVs,as assessed by morphology,size,protein concentration and EVs biomarkers expression.UEVs isolated from HFD were found suitable for miRNA detection with no significant differences in RNA yield,RNA distribution and miRNA concentrations when compared with UC.CONCLUSIONHFD is an easy and economic method for isolating EVs from large volumes of urine.HFD method is similar to conventional UC method in UEVs yields and is appropriate for miRNA analysis.Part 2 Detection and Assessment of PCa-related miRNAs in UEVsOBJECTIVEFind potential biomarkers for PCa by detecting some reported PCa-related miRNAs in UEVs and serum EVs.Compare miRNAs expression levels in UEVs and paired serum EVs and assess whether EVs miRNAs can be effective biomarkers for PCa.METHODSUEVs were isolated from patients with PCa,patients with benign prostate hyperplasia(BPH)and healthy individuals.Several PCa-related miRNAs were chosen from previous studies and measured in UEVs and paired serum EVs.The first cohort was contained by 21 PCa patients and 14 BPH patients.The signficantly differentially expressed miRNAs were detected in UEVs and paired serum EVs in the second cohort.Differences between groups were tested using two-tailed Student's t-test,one-way ANOVA and Welch's ANOVA followed by LSD or Games-Howell test as appropriate,while receiver-operating characteristic curves(ROC)were used for evaluating the diagnostic potential of miRNAs.RESULTSIn the first cohort,miR-107,miR-375,miR-21,miR-27a and miR-574 showed no significant difference between UEVs from PCa patients and BPH patients.Thus,miR-145,miR-1290,miR141,miR-572 were detected in the second cohort.In UEVs,miR-145 and miR-1290 is upregulated in PCa patients compared with BPH patients and healthy controls.In serum EVs,miR-572,miR-1290,miR-141 and miR-145 were significantly higher in patients with PCa than with BPH.Moreover,miRNAs levels in serum EVs were significantly higher than in UEVs.The ROC and area under ROC curve(AUC)revealed that miR-145 in UEVs combined with serum PSA(AUC:0.863)or miR-145 in serum EVs combined with serum PSA(AUC:0.895)could differentiate PCa from BPH better than PSA alone(AUC 0.805).In addition,significant increase was observed in UEVs miR-145 levels when patients with Gleason score?8 tumors compared with Gleason score?7.CONCLUSIONmiR-145 is deregulated from PCa patients compared BPH patients in UEVs and serum EVs.When combined serum PSA with UEVs miR-145 or serum EVs miR-145,the ability to discriminate PCa patients from BPH patients can be improved than PSA alone.We suggest the potential use of UEVs miR-145 as a biomarker of PCa.