Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer

doi:10.1016/j.canlet.2016.12.006

Cancer Letters

Volume 388, 1 March 2017, Pages 208-219

https://doi.org/10.1016/j.canlet.2016.12.006 Get rights and content

Highlights

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A series of circRNAs are differentially expressed in GC tissues compared with matched normal tissues.
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circPVT1 is upregulated in GC tissues and promotes GC cell proliferation by sponging members of the miR-125 family.
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circPVT1 is a prognostic marker for GC patients.

Abstract

Circular RNAs (circRNAs) comprise a novel class of widespread non-coding RNAs that may regulate gene expression in eukaryotes. However, the characterization and function of circRNAs in human cancer remain elusive. Here we identified at least 5500 distinct circRNA candidates and a series of circRNAs that are differentially expressed in gastric cancer (GC) tissues compared with matched normal tissues. We further characterized one circRNA derived from the PVT1 gene and termed it as circPVT1. The expression of circPVT1 is often upregulated in GC tissues due to the amplification of its genomic locus. circPVT1 may promote cell proliferation by acting as a sponge for members of the miR-125 family. The level of circPVT1 was observed as an independent prognostic marker for overall survival and disease-free survival of patients with GC. Our findings suggest that circPVT1 is a novel proliferative factor and prognostic marker in GC.

Introduction

Stomach adenocarcinoma, otherwise known as gastric cancer (GC), is one of the most common cancers worldwide and is thus a global cancer burden. According to the GLOBOCAN database, GC is the fourth most common cancer and the third-leading cause of cancer-related deaths worldwide [1]. Despite many advances in the diagnosis and treatment of this disease, the prognosis of patients with GC remains poor, with a 5-year overall survival of less than 30% in most countries [2]. Therefore, the discovery of new molecular mechanisms and therapeutic targets that may control the severity of GC and present a predictive value for prognosis are of great importance.

Circular RNAs (circRNAs) from back-spliced exons have been recently identified as a naturally occurring family of noncoding RNAs (ncRNAs) that is highly prevalent in the eukaryotic transcriptome [3], [4], [5], [6]. circRNAs are characterized by a covalently closed loop structure with neither a 5′ cap nor a 3′ polyadenylated tail. They are highly stable, are found predominantly in the cytoplasm and can be sorted into exosomes [7]. The formation of circRNA involves the occurrence of back-splicing by the canonical spliceosome [8]. Such circularization is facilitated by surrounding complementary sequences and is regulated by specific RNA binding proteins [9], [10], [11], [12], [13]. It is becoming increasingly evident that circRNAs are not simply by-products of mis-spliced RNAs or splicing errors, but rather, they are the products of regulated back-spliced RNAs with distinct sets of cis-elements and/or trans-factors [14]. Accordingly, many circRNAs have been found to be upregulated during brain development and during epithelial–mesenchymal transition (EMT) in humans [15], [16], [13]. Recently, circRNAs were shown to act as miRNA sponges that regulate gene expression [4], [17]. Importantly, a growing number of studies have demonstrated that circRNAs are closely associated with disease and may play a significant role in the pathogenesis and diagnosis of disease [18], [19], [20]. However, the characterization and function of circRNAs in human cancer remain largely unknown.

In this study, we generated ribo-minus RNA sequencing data from gastric cancer tissues of three patients, and identified approximately 5500 circRNA candidates (at least two unique back-spliced reads). We characterized one circRNA derived from the PVT1 gene locus, termed circPVT1, which is frequently upregulated in patients with GC. Functional assays revealed that circPVT1 could promote cell proliferation by sponging members of miR-125 family. circPVT1 was further found to be an independent prognostic marker for survival in patients with gastric cancer.

Section snippets

Human samples

We retrospectively collected paired cancer specimens and adjacent normal tissues from patients with gastric cancer who had surgically proven primary GC and who received a D2 radical gastrectomy (R0 resection) at Fudan University Shanghai Cancer Center (FUSCC) between December 2007 and December 2010. None of these patients received preoperative chemotherapy or radiotherapy. Clinic pathological features, which included age, gender, tumor site, tumor size, differentiation grade, TNM stage

Identification of circular RNAs by RNA-seq analysis in gastric cancer

We first characterized circular RNA transcripts using RNA-seq analysis of ribosomal RNA-depleted total RNA from three paired normal and cancerous gastric tissues (Fig. 1A). Each sample was sequenced on an Illumina HiSeq and yielded ∼60 million reads, which were mapped to the human reference genome (GRCh37/hg19) by TopHat2 [21]. A computational pipeline based on the anchor alignment of unmapped reads was used to identify circRNAs without reliance on gene annotations [4]. In all, 15623 distinct

Discussion

In this study, we identified a large number of circular RNAs in gastric cancer tissues. We also found that a substantial fraction of circRNAs were differentially expressed in cancer tissues compared with normal tissues, which suggests that these RNAs may be regulated and may exert a potential function. We further characterized one of the most differentially expressed circRNAs and provided the first evidence that a specific circRNA may play an important role as a prognostic marker in cases of

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (81472617, 81302103, 81272729 and 81502430) and Shanghai Pujiang Program (14PJ1401900). We thank members of our laboratory for providing constructive advice during the process of study design and data analysis.

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¹: These authors contributed equally to this work.

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Original ArticleCircular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer

Highlights

Abstract

Introduction

Section snippets

Human samples

Identification of circular RNAs by RNA-seq analysis in gastric cancer

Discussion

Acknowledgements

Lancet

Cell Rep.

Cell

Mol. Cell

Cell Rep.

Cell

Mol. Cell

J. Biol. Chem.

Global cancer statistics, 2012

CA: Cancer J. Clin.

circBase: a database for circular RNAs

RNA