UROLOGICAL ONCOLOGY A Significant Upregulation of miR-886-5p in High Grade and Invasive Bladder Tumors Atefeh Khoshnevisan,1 Mahmoud Parvin,2 Nasim Ghorbanmehr,3 Nasim Hatefi,2 Hamid Galehdari,1 Seyed Amir Mohsen Ziaee,4* Seyed Javad Mowla5 Purpose: To investigate the expression alteration of miR-886-5p in bladder tumors and evaluating its expression level as a potential biomarker in this type of cancer. Materials and Methods: Formalin-fixed paraffin-embedded (FFPE) samples of bladder tumors belonging to 70 patients whom had been referred to the Shahid Labbafi-Nejad medical center were obtained from the archi- val collection of pathology department. After RNA extraction and cDNA synthesis, expression levels of miR- 886-5p were quantified by a real-time reverse transcription polymerase chain reaction (RT-PCR) approach. Results: Our data revealed a significant upregulation (~3 times) of miR-886-5p in high grade bladder tumors, com- pared to the low grade ones (P < .05). Moreover, its expression level could significantly discriminate noninvasive (Ta, T1) from invasive (T2-T4) tumor stages. Conclusion: Our data suggests a potential role for miR-886-5p in progression of bladder cancer. Keywords: carcinoma; transitional cell; gene expression regulation; microRNAs; genetics; urinary bladder neo- plasms. INTRODUCTION Bladder cancer is the most common form of malig-nancy in the urinary tract, however, its molecular pathogenesis is incompletely understood.(1) It develops in a multistep process with a variety of distinct biolog- ical and functional features.(2) Conventional clinical and pathological parameters are widely used to classify bladder tumors with different grades and stages, and also to predict the clinical outcome of the disease. Neverthe- less, the predictive ability of these parameters is limited. (3) Therefore, there has been a great effort in the field to discover novel molecular pathways involved in bladder cancer, to improve its diagnosis, prognosis and treatment. microRNAs (miRNA) are small (~19-25 nucleotides) single-stranded RNA molecules, with an important role in post-transcriptional regulation of their targets via repressing gene translation or degrading target mRNAs. miRNAs are involved in development as well as in progression of a number of human cancers, including bladder cancer.(4-10) Based on a vast number of profiling experiments, the miRNA signatures are tumor type- and tissue-specific. Moreover, the signa- ture is often related to the grade and stage of the tu- mors. Thus, miRNA expression analysis could be used to classify tumors according to their grades of malignancies. It has been recently proposed that the combined expression of stem cell associated factors and specific oncogenes could induce a non-differenti- ated state in cancer cells which can then progress into high-grade ones.(11-13) Strikingly, histologically poorly differentiated tumors display a preferentially elevated expression of genes normally enriched in embryonic stem (ES) cells.(11) These molecular markers, alone or in combination with conventional approaches, have the capacity to improve diagnosis, identifying patients who will respond to chemotherapy, and finding mo- lecular targets for novel therapeutic interventions.(14-19) Regarding the potential role of miR-886-5p in stem cell self-renewal, pluripotency and differentiation, we en- couraged to investigate its potential expression in blad- der cancer tissues, and also its potential expression al- teration in tumors with different grades of malignancies. MATERIALS AND METHODS Sample Collection and Preparation Formalin-fixed paraffin-embedded (FFPE) speci- mens of bladder cancer and associated patients› data were collected from Labbafi-Nejad hospital (Tehran, Iran). A total of 70 specimens were obtained from pa- tients who had been undergone operations between 1 Department of Genetics, Shahid Chamran University of Ahvaz, Ahvaz, Iran. 2 Department of Pathology, Labbafi-Nejad Medical Centre, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3 Department of Anatomy, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 4 Urology and Nephrology Research Center, Labbafi-Nejad Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 5 Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. *Correspondence: Urology and Nephrology Research Center, Labbafi-Nejad Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel: +98 21 2256 7222. Fax: +98 21 2256 7282. E-mail: samziaee@gmail.com. Received August 2013 & Accepted June 2014 Urological Oncology 2160 2005 and 2011. The histopathological features of the samples were re-examined and confirmed by an ex- pert pathologist (M.P.), according to the grading and TNM system for stage classification of the World Health Organization. Written informed consent had been obtained from all subjects prior to sampling. The samples with inadequate tissue size, incomplete clinicopathological information, unclear tumor cells from pathological view and multiple samples from recurrent patients were excluded from the study. The Medical Ethics Committee of Tarbiat Modares University approved the experiment design. FFPE blocks were cut into thin sections and prepared for RNA extraction. RNA Extraction Sections of 15 μm thickness were prepared from each FFPE specimen. Paraffin was removed by xy- lene (Merck KGaA, Darmstadt, Germany) treatment and tissues were washed out for three times with ab- solute ethanol (Merck KGaA, Darmstadt, Germany) to remove xylene. After drying, tissues were treated with proteinase K (Fermentas, Vilnius, Lithuania) at 56˚C for 3 hours. The homogenized tissues were then employed for total RNA extraction, which was per- formed by acid guanidinium phenol chloroform proce- dure using Trizol solution (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA purity and quantity were assessed by means of spectrophotometry (Gene Quest), where A260/A280 and 230/260 ratios were used to monitor any poten- tial contamination with genomic DNA and proteins. DNase Treatment and cDNA Synthesis To remove any possible genomic DNA contamination, total RNAs were treated with DNase I (Fermentas, London, UK) at 37ºC for 30 minutes. Reverse tran- scription (RT) reaction was performed on 2 μg of pu- rified total RNA by reverse transcriptase enzyme (Ta- kara Holdings, Kyoto, Japan), as described previously. (20) RT reactions also contained 0.15 μM stem-loop RT primer (Table 1), 3 μM random hexamer (Macro- gen Inc. Seoul, South Korea), and 1 × RT buffer (Ta- kara Holdings, Kyoto, Japan). The 10 μL reactions were then incubated at 16ºC for 30 minutes, at 42ºC for 30 minutes, and at 85ºC for 5 minutes and then held at 4ºC till being used. All RT reactions, including no-template and no-RT controls, were run in duplicate. Quantitative Real-Time Reverse Transcription Poly- merase Chain Reaction (RT-PCR) Quantitative real-time RT-PCR was performed using the ABI7500 System (Applied Biosystems, CA, USA). The relative expression of miR-886-5p was assessed in comparison to U6 snRNA, as a reference internal con- trol, using specific primers. All primers were designed as shown in Table 1. Real-time PCRs were performed in a final reaction volume of 20 μL including, 10 ng cDNA, 10 μL of SYBR Green I master mix (Takara Holdings, Kyoto, Japan), and 200 nM of forward and re- verse primers, according to the manufacturer’s instruc- tions. The PCR reactions were carried out as follows: an initial denaturation at 95ºC for 5 minutes, followed by 40 cycles of denaturation at 95ºC for 10 seconds, annealing at 60ºC for 30 seconds and extension at 72ºC for 30 seconds. Authenticity of the PCR products was examined by examining the sizes of the PCR products by polyacrylamide gel electrophoresis, as well as by inspecting the uniqueness of the products melt curves. To compensate for the inter-PCR variations, the expres- sion of the target gene was normalized of that of endoge- nous control U6 snRNA. For this analysis, the compara- tive Ct (threshold cycle number) method (ΔCt) was used. Cloning and Sequencing of the Amplicons Table 1. The sequence of primers used in this study. Name Primer Primer Sequence ( 5› to 3›) PCR Product Length (nucleotides) miR-886-5p Stem-loop (for cDNA synthesis) GTCGTATCCAGTGCAGGGTCCGAGGTATTCG CACTGGATACGACCCGCTT Forward CGGGTCGGAGTTAGCTCA 58 Reverse GTGCAGGGTCCGAGGT RNU6 Forward GAACGATACAGAGAAGATTAGC 54 Reverse GAATTTGCGTGTCATCCTTG Abbreviation: PCR, polymerase chain reaction. Variables Score Age (minimum-maximum), years 38-87 Gender, No. Male 65 Female 5 Stage, No. Ta/T1 40 T2-T4 25 Undetermined 5 Grade, No. Low grade 28 High grade 35 Undetermined 7 Surgical procedure, No. TUR-BT 48 Radical cystectomy 22 Abbreviation: TUR-BT, transurethral resection of bladder tumor. Table 2. Demographic and clinical characteristics of the patients with bladder cancer. Upregulation of miR-886-5p in Bladder Tumors-Khoshnevisan et al. Vol 12 No 03 May-June 2015 2161 The amplified product of the real-time PCR with the expected size was cloned via the InsTAcloneTM PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA). The vector was then amplified through transfor- mation into DH5α, and the isolated clones were then sent for direct sequencing (Macrogen Inc. Seoul, South Korea). Statistical Analysis The obtained data were statistically analyzed by Graph- pad software (La Jolla, California, USA, www.graph- pad.com). The data were presented as mean ± standard deviation (SD) and the student unpaired t-test was used to determine the significance of the observed differenc- es between different groups. A P value less than .05 was considered statistically significant. In addition, (ROC) Receiving Operating Characteristic curve anal- ysis, with calculation of both the area under the curve and the corresponding 95% confidence intervals (CI), was used to assess the specificity and sensitivity with which the expression level of miR-886-5p could dis- criminate between low and high grade/stage tumors. RESULTS miR-886-5p is Upregulated in High Grade Bladder Tumors To evaluate the expression alteration of miR-886-5p in different bladder tumors, we collected FFPE samples from 70 patients. The age of the patients was 38-87 years old (mean, 61 years), of whom there were 65 male and 5 female. All tumor types were transitional cell car- cinoma, from which 35 were high grade, and 28 were low grade. Total RNA extraction and real-time PCR per- formed on all samples and the authenticity of the PCR products were confirmed by direct sequencing of the PCR products. All data were normalized to the expres- sion of U6, as a house-keeping internal control. There were no non-specific products or primer-dimer peaks in melt-curve analysis by ABI-7500 PCR instrument. Analysis of gene expression among different grades of malignancies of bladder tumors revealed a significant upregulation (fold change: 2.84, P = .0187) of miR-886-5p in high grade tumors, compared to that of low-grade ones (Figure 1). miR-886-5p Expression in Cancer Tissues with Dif- ferent Stages As shown in Figure 2, there was a significant differ- ence in the expression level of miR-886-5p in tumors with different stages. Considering the invasiveness, bladder cancer can be categorized either as noninvasive (Ta/T1) or invasive tumors (T2-T4). Comparing the ex- pression level in tumors with different stages revealed that miR-886-5p is significantly upregulated (with a fold change of 4.147, P = .036) in invasive tumors. Analyzing the Validity of miR-886-5p as a Tumor Marker for Bladder Cancer We used the ROC curve analysis to estimate the sensi- tivity and specificity by which the miR-886-5p expres- sion level could discriminate between bladder tumors with different grades and stages. As depicted in Fig- ure 3, ROC curve analysis yielded an AUC (the are- as under the curve) of 0.742 (95% CI: 0.6198-0.8642) and 0.67 (95% CI: 0.535-0.799) for miR-886-5p to discriminate tumors with different stages and grades, respectively. An AUC > 0.70 indicates a good abili- ty of a marker to discriminate two groups of samples. DISCUSSION In recent years, tremendous advances have been made in the discovery of new markers associated with alterations at the molecular level of bladder cancer. The studies have shown considerable clinical relevance in different areas such as tumor classification and prognosis. There are some studies which have been focused on expression analysis of some microRNAs involved in bladder cancer progression and tumor behavior. Saito and colleagues reported that miR-127 is down regulated in bladder carcinoma.(17) Furthermore, Gottardo and colleagues(18) identified 10 up regulated miRNAs in cancer samples such as miR-185, miR-203, miR-205, miR-221 and etc. In this study, we found a significant upregulation of miR-886-5p in high grade urinary bladder cancer tis- sues, compared to the low grade ones. In addition, the expression of miR-886-5p showed a significant elevation in invasive tumors, compared to the nonin- vasive samples. Employing the ROC curve analysis, we further discovered that this microRNA could po- Figure 1. Comparing the expression level of miR-886-5p in blad- der tumors with different grades of malignancies. Figure 2. Comparing the expression of miR-886-5p in bladder tu- mors with different stages. Upregulation of miR-886-5p in Bladder Tumors-Khoshnevisan et al. Urological Oncology 2162 tentially being used as a good tumor marker to dis- criminate between high and low grade bladder cancer, as well as for discriminating invasive bladder tumors from noninvasive ones. To the best of our knowl- edge, these findings are the first report on the expres- sion of miR-886-5p in bladder cancer. Therefore, we could not compare our results with previously reported data. As another limitation to our study, we failed to collect enough normal bladder tissue to compare miR- 886-5p expression between normal and tumor tissues. miR-886-5p is known as one of the miRNAs associated with the pluripotency state of stem cells. Wilson and colleagues reported the expression of miR-886-5p in human embryonic stem cells (hESC) and induced pluri- potent stem (iPS) cells, where it is down regulated upon the induction of differentiation in both types of pluripo- tent cells.(15) As indicated by Ben-Porath and colleagues a stem cell signature is present in poorly differentiated and high grade bladder tumors.(11) According to their findings, specific transcriptional regulators which are normally active in stem cells are overexpressed in poorly differentiated tumors arising in bladder. Based on our obtained data, we hypothesized that miR-886-5p regulate some pathways involved in progression, invasion and metastasis of bladder tu- mors, probably by down regulating the expression of some mRNAs functioning in preventing these path- ways. An oncogenic role for miR-886-5p has already been provided by Li and colleagues' work in which miR-886-5p inhibits apoptosis of cervical cancer cells by down-regulating the expression of Bax.(16) There are controversies in literatures on the exact nature of miR-886-5p. In some reports it has been designated as a Vault RNA or a non-coding RNA. Stadler and col- leagues claimed that the sequence of this microRNA is a part of a longer RNA named VtRNA2.(19) Later, Lee and colleagues introduced the pre-mir-886 as a 102-nucleo- tide long RNA which is abundantly presents in the cy- toplasm of the cells with unique features which differs from those of a genuine pre-microRNAs or vault RNAs. (21) They found a much lower percentage of mature miR- 886-5p compared to its precursor form, pre-miR-886, in lung cancer cell lines. The later finding could be due to either a low rate of cleavage of pre-mir-886 into mature miR-886-5p, or that, mature miR-886-5p is only a deg- radation intermediate of pre-mir-886. They also found that pre-mir-886 is suppressed in some cancer cell lines and clinical specimens, where it functions through regulation of protein kinase RNA-activate (PKR).(21) Part of the aforementioned controversies arises from the innate differences in the strategies and methods used to amplify microRNAs, and most importantly how to discriminate mature form the precursor from. In the current study, we designed a stem-loop RT primer to specifically amplify the mature form of miR-886- 5p. The stability of the stem-loop structure of the RT primer precludes its annealing to the pri- or pre-miR- NA, due to a steric hindrance.(22,23) Despite the fact that stem-loop primers are more difficult to design,(23) they are highly specific for amplifying mature miRNAs.(24,25) The later claim was further reinforced by our direct se- quencing of the real-time PCR products which proved the specific amplification of mature miR-886-5p. Regardless of the nature of amplified product in our study, as either a genuine miRNA or a piece of a longer non-coding RNA, its differential expression in tumors with different grades and stages is of great interest. 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