Identification of novel piRNAs in bladder cancer

Abstract

PIWI-interacting RNAs (piRNAs) are a newly identified class of small non-coding RNAs that can play important roles in germline development and carcinogenesis. In this study, piRNA microarrays were used to investigate global piRNA expression in three bladder cancer tissues and their adjacent normal tissues. Using the 3' untranslated region (UTR) sequence complementarily method, we predicted the target gene of piRNA. Our results showed that the expression levels of 106 piRNAs were up-regulated and 91 were down-regulated in bladder cancer tissues, among which the fold-change of down-regulated piRNA DQ594040 associated with bladder cancer (piRABC) was the highest piRNA. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to confirm piRABC expression in paired bladder cancer tissues and their adjacent normal tissues (n = 25). Over-expression of piRABC can inhibit bladder cancer cell proliferation, colony formation, and promote cell apoptosis (all P < 0.05). Luciferase reporter gene assays indicated that piRABC could increase the luciferase activity of TNFSF4. Western blotting analyses and ELISA assays also confirmed that the expression of TNFSF4 protein was up-regulated in control subjects compared with bladder cancer subjects. In conclusion, piRABC plays a crucial role in the development of bladder cancer.

Introduction

Bladder cancer is the most common cancer in the urinary system. In bladder cancer, transitional cell carcinoma (TCC) is the most predominant histopathological type, which accounts for up to 90% of all cases. The development of bladder cancer involves multi-factors and is a multi-stage process. It had been well-established that cigarette smoking and occupational and environmental exposures are risk factors for bladder cancer [1]. At present, researchers have mainly focused on investigating the effect of non-coding small regulatory RNAs, including microRNAs (miRNAs), endogenous small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs). To date, the most widely studied non-coding small RNAs are miRNAs, which can regulate gene expression via binding to the sequence of target mRNAs [2], [3], [4]. Our previous findings found that miR-146a was associated with bladder cancer risk and prognosis [5]. Compared with miRNAs, piRNAs are far less investigated RNA molecules. Recently, piRNAs have emerged as important regulators in multiple species.

piRNAs are a class of non-coding small RNA of 24–30 nucleotides and are produced via a Dicer-independent mechanism and can bind to PIWI proteins to form complexes to induce a silencing effect [6], [7], [8], [9]. Studies in mice, Drosophila, Caenorhabditis elegans, and zebrafish have indicated that piRNAs play important roles in germline development [7], [8], [9], [10], [11], [12], [13]. However, the function of PIWI-piRNA complexes has yet not been fully understood. Some studies have suggested that piRNAs have a potential role in the regulation of transposable elements in the germline [14], [15], and the PIWI-piRNA complex has been shown to regulate histone modifications at the binding site [16]. Rajasethupathy et al. revealed that piRNAs are prevalently expressed in the brain and subsequently found that the PIWI–piRNA complex can regulate methylation of a CpG island in the promoter of CREB2 via a piRNA binding to the CREB2 transcript, thereby inducing enhanced long-term synaptic facilitation in Aplysia [17]. Taken together, piRNAs have a potential role in transposon silencing and the epigenetic and post-transcriptional regulation of gene expression [18].

piRNAs have been found to be involved in the development of cancer, such as cervical cancer [19], gastric cancer [20], [21], breast cancer [22], multiple myeloma [23], and hepatocellular carcinoma [24]. These findings indicated that piRNAs play important roles as oncogenes or tumor suppressor genes in carcinogenesis. In this study, we used a piRNA profile to explore the effects of piRNA in bladder cancer and identified a novel piRNA DQ594040 associated with bladder cancer (piRABC).