Elsevier

European Urology

Volume 63, Issue 6, June 2013, Pages 1091-1100
European Urology

Urothelial Cancer
Methylation of a Panel of MicroRNA Genes Is a Novel Biomarker for Detection of Bladder Cancer

https://doi.org/10.1016/j.eururo.2012.11.030Get rights and content

Abstract

Background

Dysregulation of microRNAs (miRNAs) has been implicated in bladder cancer (BCa), although the mechanism is not fully understood.

Objective

We aimed to explore the involvement of epigenetic alteration of miRNA expression in BCa.

Design, setting, and participants

Two BCa cell lines (T24 and UM-UC-3) were treated with 5-aza-2′-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which their miRNA expression profiles were analyzed using a TaqMan array (Life Technologies, Carlsbad, CA, USA). Bisulfite pyrosequencing was used to assess miRNA gene methylation in 5 cancer cell lines, 83 primary tumors, and 120 preoperative and 47 postoperative urine samples.

Outcome measurements and statistical analysis

Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic performance of the miRNA gene panel.

Results and limitations

Of 664 miRNAs examined, 146 were upregulated by 5-aza-dC plus PBA. CpG islands were identified in the proximal upstream of 23 miRNA genes, and 12 of those were hypermethylated in cell lines. Among them, miR-137, miR-124-2, miR-124-3, and miR-9-3 were frequently and tumor-specifically methylated in primary cancers (miR-137: 68.7%; miR-124-2: 50.6%; miR-124-3: 65.1%; miR-9-3: 45.8%). Methylation of the same four miRNAs in urine specimens enabled BCa detection with 81% sensitivity and 89% specificity; the area under the ROC curve was 0.916. Ectopic expression of silenced miRNAs in BCa cells suppressed growth and cell invasion.

Conclusions

Our results indicate that epigenetic silencing of miRNA genes may be involved in the development of BCa and that methylation of miRNA genes could be a useful biomarker for cancer detection.

Introduction

MicroRNAs (miRNAs) are a group of small noncoding RNAs that negatively regulate the translation and stability of partially complementary target mRNAs. In that way, they play important roles in a wide array of biologic processes, including cell proliferation, differentiation, and apoptosis [1]. Increasing evidence suggests that dysregulation of miRNA expression contributes to the initiation and progression of human cancer [2], [3]. Altered miRNA expression is thought to play an important role in the pathogenesis of bladder cancer (BCa) and in certain tumor phenotypes. For instance, high-grade BCa exhibits upregulation of several miRNAs, including miR-21, which suppresses p53 function [4]. In addition, miR-21-to-miR-205 expression ratios are elevated in invasive BCa cells [5], while miR-200 family members regulate epithelial-to-mesenchymal transition by targeting transcription repressors ZEB1 and ZEB2 in BCa cells [6].

Although the mechanisms underlying miRNA dysregulation in cancer are not yet fully understood, recent studies have shown that the silencing of several miRNAs is tightly linked to epigenetic mechanisms, including histone modification and DNA methylation [7], [8]. For example, treatment with a histone deacetylase (HDAC) inhibitor and a DNA methyltransferase (DNMT) inhibitor restored expression of various miRNAs in cancer cells [7], [9], and the list of miRNA genes methylated in cancer is rapidly growing [10]. Studies have also shown that restoration of epigenetically silenced miRNAs may be an effective strategy for treating cancer and that aberrant methylation of miRNA genes could be a useful biomarker for cancer detection [10], [11]. In addition, it was recently shown that the silencing of miRNA expression in BCa is associated with DNA methylation, often involving the CpG island (CGI) or CpG shore [12], [13]. In an effort to identify novel biomarkers and treatment targets in BCa, we aimed to identify miRNAs epigenetically silenced in BCa cells by screening for miRNAs whose expression is upregulated by DNA demethylation and HDAC inhibition. We also investigated the methylation of miRNA genes in urine specimens and assessed its clinical usefulness as a biomarker for detection of BCa.

Section snippets

Cell lines and tissue samples

BCa cell lines (T24, UM-UC-3, HT-1197, HT-1376, SW780, and 5637) and a normal urothelial cell line (SV-HUC-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Supplementary Table 1). A colorectal cancer cell line HCT116 harboring genetic disruptions within the DNMT1 and DNMT3B loci (DNMTs KO) have been described previously [8]. T24 and UM-UC-3 cells were treated first with 1 μM or 0.1 μM 5-aza-2′-deoxycytidine (5-aza-dC; Sigma-Aldrich, St Louis, MO, USA) for 72 h,

Identification of epigenetically silenced microRNA genes in bladder cancer

To identify epigenetically silenced miRNAs in BCa, we performed TaqMan array analysis using two BCa cell lines (T24 and UM-UC-3) treated with 1 μM 5-aza-dC plus 3 mM PBA. Of the 664 miRNAs examined, the drug treatment induced upregulation (more than five-fold) of 208 miRNAs in T24 cells and 200 miRNAs in UM-UC-3 cells. Of those, 146 miRNAs were upregulated in both cell lines (Supplementary Fig. 2 and 3; Supplementary Table 4). We selected 23 miRNA genes that harbored CGIs in the proximal upstream

Discussion

We identified four miRNA genes (miR-137, miR-124-2, miR-124-3, and miR-9-3) that were frequently methylated in both cultured and primary BCa cells. Earlier studies have shown that these miRNAs are tumor-suppressive or tumor-related and that they are epigenetically silenced in cancers of various origins. Hypermethylation of miR-137 was first discovered in oral cancer [16] and has since been noted in other malignancies, including cancers of the colon [14] and stomach [3]. Within cancer cells,

Conclusions

We identified four miRNA genes that are frequent targets of epigenetic silencing in BCa. Although their specific functions in bladder carcinogenesis remain unknown, it is evident that restoration of these miRNAs may be an effective anticancer therapy. Furthermore, methylation of these miRNA genes in urine specimens could serve as a useful and noninvasive biomarker for accurate detection of BCa.

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