Investigative Urology

Expression of the Tumor Suppressive miRNA-23b/27b Cluster is a Good Prognostic Marker in Clear Cell Renal Cell Carcinoma Tomoaki Ishihara, Naohiko Seki, Satoru Inoguchi, Hirofumi Yoshino, Shuichi Tatarano, Yasutoshi Yamada, Toshihiko Itesako, Yusuke Goto, Rika Nishikawa, Masayuki Nakagawa and Hideki Enokida* From the Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University (TI, SI, HY, ST, YY, TI, MN, HE), Kagoshima and Department of Functional Genomics, Chiba University Graduate School of Medicine (NS, YG, RN), Chiba, Japan

Abbreviations and Acronyms ccRCC ¼ clear cell RCC EGFR ¼ epidermal growth factor receptor GEO ¼ Gene Expression Omnibus KEGG ¼ Kyoto Encyclopedia of Genes and Genomics OS ¼ overall survival RCC ¼ renal cell carcinoma VHL ¼ von Hippel-Lindau Accepted for publication July 1, 2014. Study received Kagoshima University bioethics committee approval. Supported by Ministry of Education, Sciences, Sports and Culture of Japan Grants-in-Aid for Science Research (Young B) 25430137. * Correspondence: Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan (telephone: þ8199-275-5395; FAX: þ81-99-265-9727; e-mail: [email protected]).

Purpose: We observed abnormal expression of the microRNA-23b/27b (miR23b/27b) cluster in our previous study of miRNA expression signatures. However, the relationship between aberrant miRNA expression and clear cell renal cell carcinoma is not well established. We investigated the functional significance of the miR-23b/27b cluster in clear cell renal cell carcinoma cells and evaluated these miRNAs as biomarkers to predict the risk of clear cell renal cell carcinoma. Materials and Methods: Expression levels of miR-23b and miR-27b were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. The association between miRNA expression and overall survival was estimated by the Kaplan-Meier method. Gain of function assays were performed using mature miR-23b and miR-27b in the 786-O and A498 renal cell carcinoma cell lines. Targets regulated by these miRNAs were predicted by in silico analysis. Results: Expression of the miR-23b/27b cluster was significantly decreased in clear cell renal cell carcinoma tissue specimens and associated with pathological grade and stage. Significantly shorter overall survival was observed in patients with lower expression of the miR-23b/27b cluster. Restoration of miR-23b and miR-27b significantly inhibited cancer cell proliferation, migration and invasion. Conclusions: Expression of the miR-23b/27b cluster was frequently decreased in clear cell renal cell carcinoma tissue. Reduced expression of these miRNAs increased the risk of disease progression and predicted poor survival. Thus, miR-23b and miR-27b function as tumor suppressors, targeting several oncogenic genes in clear cell renal cell carcinoma cells. Key Words: kidney; carcinoma, renal cell; MIRN23 microRNA, human; MIRN27 microRNA, human; biological markers

RCC is a disease in which cancer cells form in the kidney tubules. ccRCC is the most common subtype of RCC. Globally the incidence and mortality rates of RCC are increasing 2% to 3% per decade.1 At diagnosis about 30% of RCCs have already metastasized. The 5-year survival rate in patients with advanced stage RCC is poor at



5% to 10% due to recurrence or distant metastasis.2 Recently antiangiogenic multi-tyrosine kinase inhibitors were developed that have been widely used as first and second line treatments. However, these treatments only slightly extend progression-free survival, and relapse and metastasis eventually develop in

0022-5347/14/1926-1822/0 THE JOURNAL OF UROLOGY® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC. Vol. 192, 1822-1830, December 2014 Printed in U.S.A.


most patients.3 The molecular mechanisms of RCC recurrence, metastasis and drug resistance are not yet fully elucidated. Therefore, it is necessary to increase our understanding of the molecular pathways of RCC progression and metastasis using advanced genomic analysis techniques. The discovery of ncRNAs in the human genome was an important conceptual breakthrough in the postgenome sequencing era. Further improving our understanding of ncRNAs is needed for continued progress in cancer research. miRNAs are endogenous small ncRNA molecules 19 to 22 bases long that regulate protein coding gene expression by repressing translation or cleaving RNA transcripts in a sequence specific manner.4 Numerous studies show that miRNAs are aberrantly expressed in many human cancers and they have significant roles in the initiation, development and metastasis of those cancers.5 Moreover, normal regulatory mechanisms can be disrupted by the aberrant expression of tumor suppressive or oncogenic miRNAs in cancer cells. Therefore, identifying aberrantly expressed miRNAs is an important first step toward elucidating miRNA mediated oncogenic pathways. Accordingly we constructed miRNA expression signatures using several types of clinical specimens, including ccRCC, and investigated the specific role of miRNAs in ccRCC oncogenesis using differentially expressed miRNAs.6,7 Recently our analysis of miRNA expression signatures revealed that miRNAs in the miR-23b/27b cluster were significantly down-regulated in cancer tissue, suggesting that this miRNA cluster is a candidate tumor suppressor in ccRCC. Clustered miRNAs are located in close proximity on the human genome and 247 human miRNAs are clustered at 64 sites at inter miRNA distances of less than 5,000 bp.8 We previously reported that miRNAs in the miR-143/ 145 and miR-1/133a clusters function as tumor suppressors, targeting several oncogenic genes in ccRCC.6,9 In this study we hypothesized that the miR23b/27b cluster functions as a tumor suppressor by targeting several oncogenic genes involved in specific cancer related pathways in ccRCC. We also investigated expression of these miRNAs in ccRCC specimens and analyzed the potential association between the miR-23b/27b cluster and various clinicopathological factors in patients with ccRCC to evaluate its potential clinical applications. Elucidating molecular targets regulated by the tumor suppressive miR-23b/27b cluster would provide new insights into the potential molecular mechanisms of ccRCC oncogenesis and metastasis, and facilitate the development of novel diagnostic and therapeutic strategies for RCC treatment.


MATERIALS AND METHODS Clinical Specimens A total of 61 ccRCCs and 61 normal kidney specimens were collected from patients who underwent radical or partial nephrectomy at Kagoshima University Hospital between 2004 and 2010. Samples were processed and stored in RNAlaterÒ at e20C until RNA extraction. Samples were staged according to the General Rule for Clinical and Pathological Studies on Renal Cell Carcinoma10 based on the AJCC (American Joint Committee on Cancer)-UICC TNM classification.11 Median followup was 62 months. Our study was approved by the Kagoshima University bioethics committee. Prior written informed consent and approval were provided by each patient.

Cell Culture and RNA Extraction The human RCC cell lines 786-O and A498 (ATCCÒ) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and maintained in a humidified incubator (5% CO2) at 37C. Total RNA was extracted as previously described.12

Quantitative Real-time Reverse TranscriptasePolymerase Chain Reaction Stem-loop reverse transcriptase-polymerase chain reaction for miR-23b (P/N 000400) and miR-27b (P/N 000409, Applied BiosystemsÒ) were used to quantify miRNAs according to previously published conditions.12 To normalize data on miRNA quantification we used U6 (P/N 001973, Applied Biosystems). The D-D threshold count method was used to calculate the fold change.

Mature miRNA Transfection RCC cell lines were transfected with LipofectamineÒ RNAiMAX transfection reagent and Opti-MEMÔ with 10 nM mature miRNA molecules as previously described.12 Pre miRÔ miRNA Precursor (P/N AM17111) served as the negative control in gain of function experiments. Transfection efficiency of miRNA in cell lines was confirmed based on down-regulation of TWF1 (PTK9) mRNA after transfection with miR-1 as recommended by the manufacturer. Cells were seeded in 6-well plates at 2  105 cells per well for wound healing assay, 5  104 cells per well for MatrigelÒ invasion assay and in 96-well plates at 3,000 cells per well for XTT assay (Hoffman-La Roche, Basel, Switzerland).

Cell Proliferation, Migration and Invasion Assays Cell proliferation was determined by XTT assay according to manufacturer instructions. Cell migration activity was evaluated by wound healing assay as described previously.6 Cell invasion assay was performed using modified Boyden chambers as described previously.6 All experiments were done in triplicate.

Putative miR-23b and miR-27b Target Analysis and Expression To identify putative miR-23b and miR-27b target genes we used the TargetScan database (http://www.targetscan. org/). To identify molecular targets and signaling pathways regulated by the miR-23b/27b cluster we analyzed in silico and gene expression data using KEGG pathway categories ( in GeneCodis



( Gene expression data were evaluated using the GEO database (GEO accession Nos. GSE36895 and GSE22541, geo/).

Statistical Analysis Relationships between 2 or 3 variables and between numerical values were analyzed using the Mann-Whitney U or Bonferroni adjusted Mann-Whitney U test. The relationship between miR-23b and miR-27b miRNA expression was analyzed by the Spearman rank correlation. Expert StatViewÒ, version 4 was used for these analyses. The chi-square test was applied to evaluate correlations of miR-23b and miR-27b expression with clinicopathological factors. We evaluated OS in 61 patients with ccRCC using the Kaplan-Meier method. Patients were divided into 2 groups by median miR-23b and miR-27b expression, and differences were determined by the log rank test.

RESULTS miR-23b/27b Cluster Expression in ccRCC clinical specimens and cell lines.

We first evaluated miR-23b and miR-27b expression in ccRCC tissue and normal kidney tissue from 42 males and 19 females. Median age was 69 years (range 41 to 91). Table 1 lists clinicopathological characteristics. Expression levels of miR-23b and miR-27b were significantly decreased in tumor tissue compared to corresponding noncancerous tissue (each miRNA p

27b cluster is a good prognostic marker in clear cell renal cell carcinoma.

We observed abnormal expression of the microRNA-23b/27b (miR-23b/27b) cluster in our previous study of miRNA expression signatures. However, the relat...
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