Gene 556 (2015) 227–234 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Genome-wide analysis of long noncoding RNA signature in human colorectal cancer Yao Xue a,b,1,GaoxiangMaa,b,1,DongyingGuc,1, Lingjun Zhu d,QiuhanHuaa,b, Mulong Du a,b,HaiyanChua, Na Tong a, Jinfei Chen c, Zhengdong Zhang a,b,⁎, Meilin Wang a,b,⁎ a Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Nanjing Medical University, Nanjing, China b Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China c Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China d Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China article info abstract Article history: Long noncoding RNAs (lncRNAs) have been widely regarded as crucial regulators in various biological processes Received 21 October 2014 involved in carcinogenesis. However, the comprehensive lncRNA expression signature in colorectal cancer re- Accepted 28 November 2014 mains fully unknown. We performed a high throughput microarray assay to detect lncRNA expression profile Available online 29 November 2014 in three paired human colorectal cancer tissues and their adjacent normal tissues. Additional 90 paired colorectal samples were collected to verify differently expression levels of two selected lncRNAs using q-RT-PCR assay. Keywords: Bioinformatic approaches were performed to explore into the functions of these differently expressed lncRNAs. lncRNA Colorectal cancer Microarray assay showed a series of lncRNAs were differently expressed in colorectal cancer. Two of the lncRNAs, Expression profile HOTAIR and a novel lncRNA, lncRNA-422 were confirmed in more samples (P = 0.015 for HOTAIR and P =0.027 GSEA for lncRNA-422, respectively). GSEA indicated that gene sets most correlated with them were those named up- GO regulated in KRAS-over, down-regulated in JAK2-knockout, down-regulated in PDGF-over and down-regulated in TBK1-knockout, all of which were cancer-related. Subsequently, GO analyses of most significantly correlated coding genes of HOTAIR and lncRNA-422 showed that these two lncRNAs may participate in carcinogenesis by regulating protein coding genes involved in special biological process relevant to cancer. Our study demonstrated that different lncRNA expression patterns were involved in colorectal cancer. Besides, HOTAIR and lncRNA-422 were identified to participate in colorectal cancer. Further studies into biological mechanisms of differently expressed lncRNAs identified in our study will help to provide new perspective in colorectal cancer pathogenesis. © 2014 Elsevier B.V. All rights reserved. 1. Introduction noncoding transcripts (ncRNAs) (Mattick, 2004). This has been the most surprising findings of the human genome project and a remark- Over the past decades, high-throughput genomic platforms have able challenge and completion of central dogma, which was centered revealed that numerous sites of human genome are transcribed to around protein coding genes and merely regard RNA as the information transmitters. Researchers have found that only approximately 1.5% of the whole genome was responsible for protein coding (Wang and Abbreviations: MALAT1, metastasis associated lung adenocarcinoma transcript 1; Chang, 2011). In addition to the huge number, relatively conservative HOTAIR, HOX transcript antisense RNA; ANRIL, CDKN2B antisense RNA 1; PCGEM1, prostate part of the sequence and the specific temporal and spatial expression cancer (PCa) gene expression marker 1; GAPDH, glyceraldehyde 3-phosphate dehydroge- patterns also provided support for the important functions of ncRNA, nase; GSEA, gene set enrichment analysis; ES, enrichment score; GO, gene ontology; DAVID, database for annotation, visualization and integrated discovery; GOEAST, Gene which were initially proverbial “dark matter” of the genome (Ponting Ontology Enrichment Analysis Software Toolkit; KRAS, Kirsten rat sarcoma viral oncogene and Belgard, 2010). Indeed, emerging studies have demonstrated the homolog;JAK2,Januskinase2;PDGF,plateletderivedgrowthfactor;TBK1,TANK-bindingki- major biological role of ncRNA in a variety of process impacting evolu- nase1;SNHG1, small nucleolar RNA host gene1; NEAT1, nuclear paraspeckle assemblytran- tion, embryonic development, metabolism, oncogenesis, etc. (Ponting script 1; PRSS3, protease, serine, 3; MMP10, matrix metallopeptidase 10; CXCL2, chemokine (C-X-C motif) ligand 2; TP53, tumor protein p53; E2F3,E2Ftranscriptionfactor3;LEF1,lym- et al., 2009; Wilusz et al., 2009). phoid enhancer-binding factor 1. NcRNAs are roughly separated into two groups according to ⁎ Corresponding authors at: Department of Environmental Genomics, School of Public the transcript length, which are small ncRNAs and long ncRNAs Health, Nanjing Medical University, 818 Tianyuan Road, Jiangning District, Nanjing (lncRNAs) (Mattick, 2001). The former one was defined as ncRNAs 211166, China. with number of nucleotides (nt) less than 200, and was represented E-mail addresses: [email protected] (Z. Zhang), [email protected] (M. Wang). by the widely-explored microRNA, which negatively regulated the 1 These authors contribute equally to this work. expression level of protein coding genes by base pairing with their http://dx.doi.org/10.1016/j.gene.2014.11.060 0378-1119/© 2014 Elsevier B.V. All rights reserved. 228 Y. Xue et al. / Gene 556 (2015) 227–234 targets (Bartel, 2009). In contrast, another transcriptional class, role in the biological process of colorectal cancer (Ge et al., 2013; Qi lncRNAs, was described to be longer than 200 nt (Mercer et al., et al., 2013; Kogo et al., 2011). However, comprehensive studies into 2009), and a rising number of studies reported their functional role the special expression patterns of lncRNAs in colorectal cancer have as regulatory RNAs (Wilusz et al., 2009; Wapinski and Chang, 2011; not been reported. Whitehead et al., 2009). Different from the relatively accordant To investigate the potential role of lncRNA in carcinogenesis of mechanisms of microRNA regulation, functions of lncRNA cannot be colorectal cancer in a more comprehensive way, we performed a accurately forecasted by its sequence because various mechanisms microarray analysis to identify the genome-wide different expres- of lncRNAs have been identified, e.g. genomic imprinting (Lee and sion profiles of lncRNAs and mRNAs between 3 pairs of colorectal Bartolomei, 2013), chromatin modification (Marchese and Huarte, cancer tissues and their adjacent normal specimens. We further val- 2014), and post-transcriptional processing (Yoon et al., 2013). Based idated the different expression levels of two lncRNAs in more tissue on the remarkable regulatory functions of lncRNAs in multiple key samples and predicted their putative functions using bioinformatic biological processes, accumulating researches have explored into approaches. their role in human disease (Li et al., 2013). It is well established that a series of lncRNAs were dysregulated in diseases, especially malignant tumors, such as MALAT1 (Ji et al., 2003), HOTAIR (Gupta et al., 2010), 2. Material and methods ANRIL (Cunnington et al., 2010), PCGEM1 (Petrovics et al., 2004), etc. Besides, the observed abnormal expressions of lncRNAs were also 2.1. Patient specimens and clinical assessments important indications of their biological functions in carcinogenesis. Although aberrant expression of lncRNAs has been gradually recog- The present study recruited 98 pairs of colorectal cancer tissue nized as a biological signature of cancers (Gibb et al., 2011), studies and corresponding non-tumor tissue samples, all of which were obtain- into the detailed expression pattern of lncRNAs in a special tumor ed from patients who underwent surgical operation at the First Affiliat- were still deficient. ed Hospital and Nanjing First Hospital of Nanjing Medical University Colorectal cancer is the third most common cancer and the fourth from September 2010. All the participants were histologically con- leading cause of cancer related death worldwide (Jemal et al., 2011), firmed to be colorectal adenocarcinoma and did not receive any other with more than one million new cases diagnosed each year (Karsa forms of therapy on the time of enrollment. Clinical information for all et al., 2010). Due to the changes of human living habits, the prevalence the subjects was obtained retrospectively from clinical files. Tumor rate of colorectal cancer has dramatically risen in China (Sung et al., grade of colorectal cancer was divided into low, intermediate, and 2005). Nowadays, colorectal cancer has been a great threat to public high, while the pathological stage was classified into Dukes A, B, C, health. Therefore, a substantial amount of studies have investigated mo- and D. The detailed information of 3 samples selected to be analyzed lecular abnormalities in occurrence of colorectal cancer, in order to learn in microarray platform and the other 95 samples in verification stage more about pathogenesis of colorectal cancer (Colussi et al., 2013; are shown in Supplementary Tables 1 and 2. A questionnaire about life- Vaiopoulos et al., 2014). Among the numerous molecules demonstrated style factors was administered to all the subjects through face-to-face to be involved in colorectal cancer, lncRNAs have drawn emerging at- interviews.