OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2015.4014 OL-0-0-4014 Articles Increased oncogenic microRNA-18a expression in the peripheral blood of patients with prostate cancer: A potential novel non-invasive biomarker AL-KAFAJIGHADA 1 AL-NAIEBZIAD TARIQ 2 BAKHIETMOIZ 1 Department of Molecular Medicine, College of Medicine and Medical Sciences, Arabian Gulf University, Manama 329, Kingdom of Bahrain Department of Surgery, College of Medicine and Medical Sciences, Arabian Gulf University, Manama 329, Kingdom of Bahrain Correspondence to: Dr Ghada Al-Kafaji, Department of Molecular Medicine, College of Medicine and Medical Sciences, Arabian Gulf University, Salmanyia Avenue, Building 293, Road 2904, Manama 329, Kingdom of Bahrain, E-mail: ghadaa@agu.edu.bh 02 2016 09 12 2015 11 2 1201 1206 30112014 10112015 Copyright © 2016, Spandidos Publications 2016

MicroRNAs have been demonstrated to be stably detectable in peripheral blood, thus representing important sources of non-invasive biomarkers of various diseases, including cancer. Recently, microRNA-18a (miR-18a) has been revealed to be highly expressed in prostate cancer (PC) tissues, acting as an oncogenic miRNA. The present study evaluated miR-18a expression in the peripheral blood of patients with PC, patients with benign prostatic hyperplasia (BPH), and healthy individuals, to assess the feasibility of using peripheral blood miR-18a as a potential non-invasive biomarker for PC. Total RNA was extracted from peripheral whole blood samples from 24 PC patients, 24 BPH patients and 23 healthy control individuals. The expression of miR-18a was assessed by reverse transcription quantitative polymerase chain reaction. The results revealed that miR-18a expression was significantly higher in PC patients than in BPH patients and healthy controls [fold change (mean ± standard deviation), 5.5±1.4 for PC, 1.5±0.5 for BPH and 1.2±0.6 for controls; P<0.005]. Higher miR-18a expression was strongly associated with PC [odds ratio (OR), 4.602; 95% confidence interval (CI), 2.194–9.654; P=0.001], but was not significantly associated with BPH (OR, 1.2; 95% CI, 0.7–2.02; P=0.332). Despite the small number of patients, which limits the statistical power of the study, higher miR-18a expression was observed to be significantly correlated with certain clinicopathological parameters, including Gleason score >7 and pathological tumor stage 3/4 (P<0.005). A receiver operating characteristic (ROC) analysis revealed that miR-18a discriminated PC patients from BPH patients and healthy controls [area under the curve (AUC), 0.805; 95% CI, 0.704–0.906). Furthermore, use of the ROC curve to discriminate PC from BPH patients yielded an AUC of 0.878 (95% CI, 0.783–0.972). In summary, the present results indicate that miR-18a expression is significantly increased in peripheral blood of patients with PC compared with that of BPH patients and healthy individuals, and that higher miR-18a expression is associated with progression of PC. Peripheral blood oncogenic miR-18a may serve as a potential novel non-invasive biomarker for PC that also facilitates discrimination between PC and BPH.

 prostate cancer benign prostatic hyperplasia microRNA biomarker peripheral blood miR-18 Introduction

Prostate cancer (PC) is the most widely diagnosed malignant tumor and the second leading cause of cancer-associated mortality in males (1). The mechanisms underlying the occurrence and progression of the disease remain largely unknown. Generally, patients with PC do not exhibit symptoms until the disease becomes locally advanced or metastatic (2). Prostate-specific antigen (PSA) detection in serum is the current standard diagnostic tool for PC. In healthy males, PSA is present in small quantities in the serum (typically <4 ng/ml), while those with PC generally present with a higher PSA (3). However, serum PSA has been demonstrated to be elevated in various other conditions, including urinary retention and prostatitis, and following trauma or physical manipulation (3). Furthermore, serum PSA may be increased in patients with benign prostate hyperplasia (BPH), which often leads to unnecessary biopsies (4). The development of more effective non-invasive biomarkers would be clinically significant for the detection of PC, and also to facilitate the differentiation of PC from BPH (4,5).

MicroRNAs (miRNAs) are short non-coding RNA molecules with an average length of 22 nucleotides. They regulate gene expression through binding to target messenger RNAs (mRNAs) and promoting their degradation and/or directing post-transcriptional repression (6). miRNAs are important for a wide range of cellular functions, including cell proliferation, differentiation and apoptosis (7). Aberrant expression of miRNAs is also implicated in various diseases, including cancer (8). Analysis of miRNA expression profiles in cancer cells has revealed that dysregulation of these molecules contributes to the aberrant activation of oncogenes and the inactivation of tumor suppressor genes in human carcinogenesis (9), and several of these miRNAs have been reported to be both up- or downregulated (10,11). In particular, miRNA (miR)-18a, which is located in the potentially oncogenic miR-17-92 cluster (12,13), is highly expressed in various types of cancer (13–15). Furthermore, miR-18a has recently been demonstrated to act as an oncogenic miRNA in PC by promoting tumorigenesis in in vitro and in vivo models of PC (16).

It has been demonstrated that miRNAs are present in human peripheral blood and exhibit resistance to endogenous RNase activity (17). In addition, miRNAs circulate in the blood of patients with diseases and of healthy individuals, and are remarkably stable, making their isolation and analysis easy (17). These unique characteristics make peripheral blood miRNAs potential novel sources of non-invasive biomarkers.

Although the origin and function of blood miRNAs are unclear, it has been demonstrated that miRNAs may be packaged into lipid-based carriers, such as exosomes, microparticles or apoptotic bodies, and actively secreted into the circulation where they bind to neighboring cells to regulate the expression of target genes, thus mediating cell-to-cell communication (18).

To date, circulating miRNAs in serum and plasma have been investigated as potential diagnostic and prognostic biomarkers for a diverse range of diseases (19–21). A number of studies have also revealed that miRNAs circulating in whole blood serve as a novel class of biomarkers in various types of malignancy, including brain (22) and breast (23) cancers, as well as in non-malignant diseases (24,25). Furthermore, a recent study by Median-Villaamil et al (26) has demonstrated that specific miRNAs are differently expressed in the whole blood of PC patients compared with that in healthy individuals, suggesting their potential for use as biomarkers in the diagnosis of PC.

The aim of the present study was to evaluate miR-18a expression in the peripheral whole blood of PC and BPH patients, and in healthy individuals, in order to test the potential of blood miR-18a as non-invasive biomarker for PC.

 Materials and methods <sec> <title>Participants

The current study was approved by the Medical Research and Ethics Committee of the College of Medicine and Medical Sciences, Arabian Gulf University (Manama, Bahrain). Between June 2013 and January 2014, whole blood samples were collected at the Urological Clinic of King Abdullah Medical City (Manama, Bahrain). The samples were obtained from 24 patients with surgically treated, localized PC, one day prior to radical prostatectomy. Tumor aggressiveness was determined by histological tumor grading system in the Gleason grading (27,28). Prostatic carcinomas with final score <7 were considered low-grade, and with a final score >7 were considered high-grade. PC was staged using the TNM system, which assesses the tumor (T), lymph nodes (N) and metastases (M) (29). Blood samples were also obtained from 24 patients diagnosed with BPH by biopsy specimen of the prostate and who exhibited an increased PSA level (>4 ng/ml) or abnormal digital rectal examination findings, and from 23 healthy controls. All participants provided written informed consent prior to blood collection. As treatment may influence the level of circulating miRNAs, only blood samples from patients who received no hormone ablative or cytotoxic therapy were used.

 RNA extraction

Whole blood samples (5 ml) were collected in tubes containing EDTA. Aliquots (0.5 ml each) of EDTA blood were mixed with 1.3 ml RNAlater®, an RNA stabilization reagent (Ambion; Thermo Fisher Scientific, Austin, TX, USA). Samples were stored at −80°C until RNA extraction. Prior to RNA extraction, frozen samples were thawed at room temperature, and total RNA (including small RNA) was isolated using the miRNeasy kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's recommendations. Concentration and purity of RNA was quantified by measuring the absorbance at 260 nm (A260) and 280 nm (A280) using a Thermo Scientific™ Nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

 Reverse transcription (RT) and quantitative (q) polymerase chain reaction (PCR)

For RT, an Applied Biosystems™ TaqMan MicroRNA Reverse Transcription kit (Thermo Fisher Scientific) was used. Total RNA (20 ng) was mixed with specific stem-loop RT primers (3 µl), 100 mM dNTPs (0.15 µl), 10X RT buffer (1.5 µl), 20 U/µl RNase inhibitor (0.19 µl), 50 U/ml MultiScribe™ Reverse Transcriptase (1 µl) and nuclease-free water (4.16 µl) to a final volume of 15 µl. For synthesis of cDNA, reaction mixtures were incubated at 16°C for 30 min, at 42°C for 30 min and at 85°C for 5 min, and then held at 4°C. Subsequently, 1.33 µl of cDNA was amplified using 10 µl of TaqMan 2X Universal PCR Master Mix II (Applied Biosystems; Thermo Fisher Scientific), 1 µl of gene-specific primers and 7.67 µl of nuclease-free water in a final volume of 20 µl. qPCR was run on a 7900HT Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific), and the reaction mixtures were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The PCR primers had the following sequences: Mature hsa-miR-18a (target), 5′-UAAGGUGCAUCUAGUGCAGAUAG-3′; and RNU6B (reference), 5′-CGCAAGGATGACACGCAAATTCGTGAGCGTTCCATATTTTT-3′.

The threshold cycle (Ct) values were calculated using SDS software version 1.4 (Applied Biosystems; Thermo Fisher Scientific). The expression of miR-18a in the blood was normalized to the expression of U6 small nuclear RNA (RNU6B). The fold change in miR-18a expression was determined using the 2−ΔΔCt method (30). ΔCt was calculated by subtracting the Ct values of RNU6B from the Ct values of the miR-18a. ΔΔCt was then calculated by subtracting the ΔCt of the healthy control samples from the ΔCt of the case samples (PC and BPH).

 Statistical analysis

Statistical analyses were performed using SPSS software version 19.0 (IBM SPSS, Armonk, NY, USA). Comparisons between cases and controls were analyzed by χ2 or Student's t-tests, and data were presented as the mean ± standard deviation (SD). Multivariate logistic regression analysis was used to assess the association of miR-18a expression with PC and BPH. Receiver operating characteristic (ROC) curves were used to assess miR-18a as a biomarker, and the area under the curve (AUC) was reported. P<0.05 (two-tailed) was considered to indicate a statistically significant difference.

 Results <sec> <title>Patient characteristics

The demographics and clinical characteristics of the participants are outlined in Table I. No significant differences in age were observed between PC patients, BPH patients and healthy control individuals (P>0.005). Serum PSA levels were significantly higher in PC patients than in healthy control individuals (P<0.005), and were significantly increased in BPH patients compared with that of healthy controls (P<0.005).

 miR-18a expression in PC, BPH and healthy controls

The expression of miR-18a was determined relative to the endogenous control, RNU6B, in the peripheral blood of PC and BPH patients and healthy control subjects using qPCR. Values were expressed as the mean (± SD) fold difference in gene expression. miR-18a and RNU6B yielded reliable Ct values in all samples from PC, BPH and control subjects. No replicates with Ct >35 were detected.

The expression of miR-18a normalized to that of RNU6B was significantly increased in the peripheral blood of patients with PC compared with that of BPH patients and healthy control individuals (P<0.005) (Fig. 1). The mean relative expression of miR-18a was 5.5±1.4 for PC patients, 1.5±0.5 for BPH patients and 1.2±0.6 for healthy controls. Multivariate logistic regression analysis revealed a strong association between higher miR-18a expression and PC [odds ratio (OR), 4.602; 95% confidence interval (CI), 2.194–9.654; P=0.001). By contrast, higher miR-18a was not significantly associated with BPH (OR, 1.2; 95% CI, 0.7–2.02; P=0.332).

 Association between miR-18a expression and pathological variables

PC patients with more aggressive tumors exhibited significantly higher miR-18a expression compared with that of patients with less aggressive tumors. The mean relative miR-18a expression in the group of patients with a Gleason score <7 was 2.4±0.4, and increased progressively to 4.4±0.3 in patients with a Gleason score of 7, and to 8.6±0.8 in patients with a Gleason score >7 (Fig. 2A). In addition, miR-18a expression was significantly higher in patients at an advanced stage of disease: Patients with a pathological tumor (pT) stage of 3 or 4 exhibited a mean relative miR-18a expression level of 8.2±1.4, which was significantly increased compared with patients of pT stage 1 or 2, in whom a mean relative expression level of 3.6±1.0 was observed (P<0.005) (Fig. 2B).

 miR-18a expression as a diagnostic tool

As miR-18a was differentially expressed in the peripheral blood of PC patients, BPH patients and control subjects, the potential of peripheral blood oncogenic miR-18a as a biomarker was evaluated. Based on an ROC analysis, blood miR-18a was able to distinguish PC patients from BPH patients and controls (AUC, 0.805; 95% CI, 0.704–0.906; P<0.001; Fig. 3A). Furthermore, miR-18a was able to distinguish PC patients from BPH patients (AUC, 0.878; 95% CI, 0.783–0.972; P<0.001; Fig. 3B).

 Discussion

miRNAs are part of a class of small non-coding RNAs that function as translational repressors by partially pairing to the 3′ untranslated region of target mRNAs (7). miRNAs are involved in highly regulated processes, including proliferation, differentiation and apoptosis (7), and are also implicated in a range of diseases (8). Notably, miRNAs serve significant roles in cancer owing to their ability to function as regulators of tumor suppressors and oncogenes (9).

Circulating tumor-derived miRNAs were first described in peripheral blood by Mitchell et al (17), who found that circulating miRNAs had the potential to be novel biomarkers in patients with PC. The authors showed that tumor-derived miRNAs can enter the circulation even when originating from an epithelial cancer type. They also showed that circulating miRNAs in plasma remained stable even after prolonged room temperature incubation or cycles of freezing-thawing. Subsequently, circulating miRNAs in the serum, plasma and whole blood have been reported as biomarkers in various different types of cancer, in addition to other diseases (19–26).

The present study evaluated the levels of miR-18a, a highly expressed miRNA in the tissues of PC (16), in the peripheral blood of patients with PC or BPH, and in healthy individuals, to test the feasibility of using peripheral blood miR-18a as a potential novel non-invasive biomarker for PC.

miR-18a is expressed as part of the miR-17-92 cluster, which comprises seven miRNAs: miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a. This cluster has been reported to have potential oncogenic functions in tumorigenesis and cancer development (12,13). In particular, miR-18a has been demonstrated to be significantly elevated in various types of cancer, including gastric cancer (14) and urothelial carcinomas of the bladder (15).

Recently, miR-18a has been reported to exhibit oncogenic activity in PC by targeting serine/threonine-protein kinase 4 (STK4) (16). Upregulation of miR-18a is capable of inducing a decrease in the STK4 protein level, and an increase in AKT phosphorylation to enhance tumor cell survival. By contrast, miR-18a knockdown in nude mice was demonstrated to decrease prostate tumor growth and induce apoptosis (16). STK4 is a proapoptotic protein that was identified as a tumor suppressor in cancer cells (31,32). Therefore, inhibition of miR-18a expression was suggested as a therapeutically beneficial strategy for the treatment of PC (16).

The results of the current study demonstrated that the expression of peripheral blood miR-18a was significantly higher in patients with PC than in BPH patients and healthy control individuals. Furthermore, a strong association was observed between higher miR-18a expression and PC. In addition, significantly higher expression of blood miR-18a was detected in PC patients with more aggressive tumors (Gleason score >7) and of a more advanced stage (pT3/4), suggesting that miR-18a expression may correlate with progression of the disease.

Increased expression of circulating miR-18a has been previously reported in the plasma of patients with pancreatic cancer (33), and higher plasma miR-18 expression has also been observed in patients with colorectal cancer (34). Furthermore, circulating miR-18a has been demonstrated to be a promising biomarker for the diagnosis and monitoring of esophageal squamous cell carcinoma (35).

In the current study, an ROC analysis revealed that peripheral blood miR-18a was able to discriminate patients with PC from BPH patients and healthy controls. Notably, peripheral blood miR-18a was additionally able to differentiate between patients with PC and patients with BPH.

PC is the most frequently diagnosed malignancy and the second major cause of cancer-associated mortality among males (1). BPH is commonly misdiagnosed as PC (36), and this frequently leads to unnecessary biopsies (3). The evaluation of non-invasive biomarkers in peripheral blood could aid in the early detection of PC, and also facilitate the discrimination between patients with malignant and benign prostatic tumors, thereby avoiding the need for invasive biopsies. Such biomarkers may also be important in the future treatment of this cancer.

A limitation of the present study is the small sample size, which may restrict the statistical power of the study. Therefore, the potential of peripheral blood miR-18a expression as a biomarker for PC must be confirmed and validated in larger populations.

In conclusion, the current results demonstrated that expression of miR-18a is increased in the peripheral blood of patients with PC compared with BPH patients and healthy controls, and that higher miR-18a expression is correlated with PC progression. Thus, peripheral blood oncogenic miR-18a may offer potential for utilization as a novel non-invasive biomarker for PC that also allows discrimination between PC and BPH.

 Acknowledgements

This work was supported by a research grant from the College of Medicine and Medical Sciences at Arabian Gulf University (grant no. 93).

 References JemalABrayFCenterMMFerlayJWardEFormanDGlobal cancer statisticsCA Cancer J Clin616990201110.3322/caac.2010721296855 MettlinCLeeFDragoJMurphyGPThe American Cancer Society National Prostate Cancer Detection Project. HistopathologyCancer67294958199110.1002/1097-0142(19910615)67:12<2949::AID-CNCR2820671202>3.0.CO;2-X1710531 NadlerRBHumphreyPASmithDSCatalonaWJRatliffTLEffect of inflammation and benign prostatic hyperplasia on elevated serum prostate specific antigen levelsJ Urol154407413199510.1016/S0022-5347(01)67064-27541857 BrawerMKProstate-specific antigen: C urrent statusCA Cancer J Clin49264281199910.3322/canjclin.49.5.26411198954 GretzerMBPartinAWPSA markers in prostate cancer detectionUrol Clin North Am30677686200310.1016/S0094-0143(03)00057-014680307 BartelDPMicroRNAs: Genomics biogenesis, mechanism and functionCell116281297200410.1016/S0092-8674(04)00045-514744438 KloostermanWPPlasterkRHThe diverse functions of microRNAs in animal development and diseaseDev Cell11441450200610.1016/j.devcel.2006.09.00917011485 HaTYMicroRNAs inH uman diseases: From cancer to cardiovascular diseaseImmune Netw11135154201110.4110/in.2011.11.3.13521860607 KentOAMendellJTA small piece in the cancer puzzle: MicroRNAs as tumor suppressors and oncogenesOncogene2561886196200610.1038/sj.onc.120991317028598 LuJGetzGMiskaEAAlvarez-SaavedraELambJPeckDSweet-CorderoAEbertBLMakRHFerrandoAAMicroRNA expression profiles classify human cancersNature435834838200510.1038/nature0370215944708 OzenMCreightonCJOzdemirMIttmannMWidespread deregulation of microRNA expression in human prostate cancerOncogene2717881793200810.1038/sj.onc.121080917891175 DewsMHomayouniAYuDAugmentation of tumor angiogenesis by a Myc activated microRNA clusterNat Genet3810601065200610.1038/ng185516878133 HayashitaYOsadaHTatematsuYA polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferationCancer Res6596289632200510.1158/0008-5472.CAN-05-235216266980 YaoYSuoALLiZFMicroRNA profiling of human gastric cancerMol Med Rep2963970200921475928 de la Ayala PeñaFKanasakiKKanasakiMTangiralaNMaedaGKalluriRLoss of p53 and acquisition of angiogenic microRNA profile are insufficient to facilitate progression of bladder urothelial carcinoma in situ to invasive carcinomaJ Biol Chem2862077820787201110.1074/jbc.M110.19806921388952 HsuTIHsuCHLeeKHMicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivoOncogenesis3e99201410.1038/oncsis.2014.1224752237 MitchellPSParkinRKKrohEMCirculating microRNAs as stable blood-based markers for cancer detectionProc Natl Acad Sci USA1051051310518200810.1073/pnas.080454910518663219 KosakaNIguchiHYoshiokaYTakeshitaFMatsukiYOchiyaTSecretory mechanisms and intercellular transfer of microRNAs in living cellsJ Biol Chem2851744217452201010.1074/jbc.M110.10782120353945 ZhengHZhangLZhaoYPlasma miRNAs as diagnostic and prognostic biomarkers for ovarian cancerPLoS One8e77853201310.1371/journal.pone.007785324223734 ChenXBaYMaLCharacterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseasesCell Res189971006200810.1038/cr.2008.28218766170 WangGKZhuJQZhangJTCirculating microRNA: A novel potential biomarker for early diagnosis of acute myocardial infarction in humansEur Heart J31659666201010.1093/eurheartj/ehq01320159880 RothPWischhusenJHappoldCChandranPAHoferSEiseleGWellerMKellerAA specific miRNA signature in the peripheral blood of glioblastoma patientsJ Neurochem118449457201110.1111/j.1471-4159.2011.07307.x21561454 SchrauderMGStrickRSchulz-WendtlandRStrisselPLKahmannLLoehbergCRLuxMPJudSMHartmannAHeinACirculating micro-RNAs as potential blood-based markers for early stage breast cancer detectionPLoS One7e29770201210.1371/journal.pone.002977022242178 MederBKellerAVogelBHaasJSedaghat-HamedaniFKayvanpourEJustSBorriesARudloffJLeidingerPMicroRNA signatures in total peripheral blood as novel biomarkers for acute myocardial infarctionBasic Res Cardiol1061323201110.1007/s00395-010-0123-220886220 KellerALeidingerPBauerAElsharawyAHaasJBackesCWendschlagAGieseNTjadenCOttKToward the blood-borne miRNome of human diseasesNat Methods8841843201110.1038/nmeth.168221892151 Median-VillaamilVMartinez-BreijoSPortela-PereiraPQuindós-VarelaMSantamarina-CainzosIAntón-AparicioLMGómez-VeigaFCirculating MicroRNAs in blood of patients with prostate cancerActas Urol Esp386336392014(In English, Spanish)10.1016/j.acuroe.2014.10.00224661838 GleasonDFMellingerGTPrediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical stagingJ Urol111586419744813554 GleasonDFHistologic grading of prostate cancer: A perspectiveHum Pathol23273279199210.1016/0046-8177(92)90108-F1555838 SobinLHWittekindCHTNM Classification of Malignant Tumours6thNew York, NY, USAWiley-Liss1841872002 SchmittgenTDLivakKJAnalyzing real-time PCR data by the comparative C(T) methodNat Protoc311011108200810.1038/nprot.2008.7318546601 MinooPZlobecIBakerKTornilloLTerraccianoLJassJPLugliAPrognostic significance of mammalian sterile20-like kinase 1 in colorectal cancerMod Pathol20331338200710.1038/modpathol.380074017277767 ZhouDConradCXiaFParkJPayerBYinYLauwersGYThaslerWLeeJTAyruchJBardeesyNMst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogeneCancer Cell16425438200910.1016/j.ccr.2009.09.02619878874 MorimuraRKomatsuSIchikawaDNovel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancerBr J Cancer10517331740201110.1038/bjc.2011.45322045190 LuoXStockCBurwinkelBBrennerHIdentification and evaluation of plasma MicroRNAs for early detection of colorectal cancerPLoS One8e62880201310.1371/journal.pone.006288023690963 HirajimaSKomatsuSIchikawaDTakeshitaHKonishiHShiozakiAMorimuraRTsujiuraMNagataHKawaguchiTClinical impact of circulating miR-18a in plasma of patients with oesophageal squamous cell carcinomaBr J Cancer10818221829201310.1038/bjc.2013.14823579215 HoffmanRMGillilandFDAdams-CameronMHuntWCKeyCRProstate-specific antigen testing accuracy in community practiceBMC Fam Pract319200210.1186/1471-2296-3-1912398793

miR-18a expression in peripheral blood of PC patients, BPH patients and healthy individuals. The expression of miR-18a relative to that of U6 small nuclear RNA was determined by quantitative polymerase chain reaction in peripheral blood samples from PC patients (n=24), BPH patients (n=24) and healthy control individuals (n=23). Data are presented as the mean ± standard deviation. *P<0.05 vs. BPH patients and controls. miR-18a, microRNA-18a; PC, prostate cancer; BPH, benign prostatic hyperplasia.

Association between pathological parameters and miR-18a expression in peripheral blood of prostate cancer patients. (A) Relative miR-18a expression in patients with Gs <7 (n=5), Gs =7 (n=10) and Gs >7 (n=9) (*P<0.05 vs. Gs =7 and <7). (B) Relative miR-18a expression in patients of stage pT1/2 (n=14) and pT3/4 (n=10) (*P<0.05 vs. pT1/2). miR-18a expression was measured relative to U6 small nuclear RNA using quantitative polymerase chain reaction, and all data are presented as the mean ± standard deviation. miR-18a, microRNA-18a; Gs, Gleason score; pT, pathological tumor stage.

ROC curve analysis (A) miR-18a is able to distinguish PC patients from BPH patients and healthy control individuals (AUC, 0.864; 95% CI, 0.751–0.977; P<0.001). (B) miR-18a can distinguish PC patients from BPH patients (AUC, 0.878; 95% CI, 0.783–0.972; P<0.001). ROC, receiver operating characteristic; miR-18a, microRNA-18a; PC, prostate cancer; BPH, benign prostatic hyperplasia; AUC, area under the curve; CI, confidence interval.

Characteristics of participants.

Variable Prostate cancer Benign prostatic hyperplasia Healthy controls
Total subjects; n 24 24 23
Age, years; mean (range) 71 (65–79) 70 (66–78) 71 (65–76)
Prostate-specific antigen, ng/ml; mean (± SD) 17.8 (±4.72) 10.5 (±2.65) 3.5 (±0.40)
Gleason score; n (%)
  <7 (low)   5 (20.8) – –
  7 (intermediate) 10 (41.7) – –
  >7 (high)   9 (37.5) – –
Tumor stage; n (%)
  pT1/2 14 (58.3) – –
  pT3/4 10 (41.7) – –
Node stage; n (%)
  N0 22 (91.6) – –
  N1 2 (8.3) – –
Metastasis stage; n (%)
  M0   24 (100.0) – –
  M1 0 (0.0) – –