INTERNATIONAL JOURNAL OF ONCOLOGY 49: 2023-2036, 2016 Integrated analysis of long non-coding RNA-associated ceRNA network reveals potential lncRNA biomarkers in human lung adenocarcinoma JING SUI*, YUN-HUI LI*, YAN-QIU ZHANG, CHENG-YUN LI, XIAN SHEN, WEN-ZHUO YAO, HUI PENG, WEI-WEI HONG, LI-HONG YIN, YUE-PU PU and GE-YU LIANG Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu 210009, P.R. China Received August 4, 2016; Accepted September 27, 2016 DOI: 10.3892/ijo.2016.3716 Abstract. Accumulating evidence has highlighted the impor- in LUAD. The present study provides novel insight for better tant roles of long non-coding RNAs (lncRNAs) acting as understanding of lncRNA-related ceRNA network in LUAD competing endogenous RNAs (ceRNAs) in tumor biology. and facilitates the identification of potential biomarkers for However, the roles of cancer specific lncRNAs in lncRNA- diagnosis and prognosis. related ceRNA network of lung adenocarcinoma (LUAD) are still unclear. In the present study, the 465 RNA sequencing Introduction profiles in LUAD patients were obtained from The Cancer Genome Atlas (TCGA) database, which provides large sample Lung cancer is the most commonly diagnosed and fatal RNA sequencing data free of charge, and 41 cancer specific cancer, which was estimated as 1.8 million new cases and lncRNAs, 25 miRNAs and 1053 mRNAs (fold change >2, almost 1.6 million deaths occurring worldwide in 2012 (1,2). P<0.05) were identified. Then, the lncRNA-miRNA-mRNA Lung adenocarcinoma (LUAD) is major pathologic subtype of ceRNA network of LUAD was constructed with 29 key lung cancer in non-smoking males, and in females (smokers lncRNAs, 24 miRNAs and 72 mRNAs. Subsequently, we or non-smokers) (3,4). Based on the GLOBOCAN series, the selected these 29 key lncRNAs to analyze their correlation with incidence of LUAD has significantly increased over the past clinical features, and 21 of them were aberrantly expressed few decades in Africa, America and Asia (5). LUAD mainly with tumor pathological stage, TNM staging system, lymph occurs in the peripheral region of the lungs, with ability to node metastasis and patient outcome assessment, respectively. metastasize to the lymph nodes and other organs. Previous Furthermore, there were 5 lncRNAs (BCRP3, LINC00472, studies have shown that several genetic abnormalities were CHIAP2, BMS1P20 and UNQ6494) positively correlated associated with the initiation and development of LUAD with overall survival (OS, log-rank P<0.05). Finally, 7 cancer (6-10), but the pathogenesis contributing to biological proper- specific lncRNAs were randomly selected to verify the expres- ties of LUAD remain inconclusive. sion in 53 newly diagnosed LUAD patients using qRT-PCR. Non-coding RNAs (ncRNAs) are a class of RNA molecules The expression results between TCGA and qRT-PCR were with no protein-coding function, which are widely expressed 100% in agreement. The correlation between AFAP1-AS1 in organisms. ncRNAs are divided into several subtypes, such and LINC00472 and clinical features were also confirmed. as microRNAs (miRNAs), small nucleolar RNA (snoRNA), Thus, our results showed the lncRNA expression profiles and small nuclear RNA (snRNA), ribosomal RNA (rRNA), we constructed an lncRNA-miRNA-mRNA ceRNA network transfer ribonucleic acidtRNA (tRNA) and long non-coding RNA (lncRNA). Recent studies indicate that ncRNAs play important biological roles in various biological processes, such as regu- Correspondence to: Professor Ge-Yu Liang, Key Laboratory late gene expression, cell proliferation, differentiation and of Environmental Medicine Engineering, Ministry of Education, apoptosis (11-15). lncRNAs, ranging from 200 nucleotides to School of Public Health, Southeast University, Nanjing, Jiangsu 100 kb in length, have become an area of increased research 210009, P.R. China (12,16). lncRNAs may regulate gene expression at the tran- E-mail: [email protected] scriptional, post-transcriptional and epigenetic levels (17-19). Because of the stronger tissue specificity, lncRNAs can be *Contributed equally more effective as early diagnosis and screening of cancer Key words: long non-coding RNAs, lung adenocarcinoma, The biomarkers (20). Increasing number of studies on lncRNAs Cancer Genome Atlas, competing endogenous RNAs network, has been performed in various types of cancer, such as competing endogenous RNAs, overall survival lung, esophageal, gastric, hepatocellular and colorectal cancer (21-25.) The aberrantly expressed lncRNAs, which 2024 SUI et al: analysis of ceRNA network reVeals biomarkers in Lung Adenocarcinoma regulate initiation, development, invasion and metastasis of are open-access and publicly available. The present study tumors have been detected in LUAD (10,26). meets the publication guidelines provided by TCGA (http:// The ceRNA (competing endogenous RNA) hypothesis cancergenome.nih.gov/publications/publicationguidelines). was proposed as a novel regulatory mechanism between In addition, 53 frozen tissue specimens (tumor tissues non-coding RNA and coding RNA (27). lncRNA can interact and their paired adjacent non-cancerous tissues) from LUAD with the miRNA by miRNA-binding sites (MREs), thereby patients collected between 2006 and 2015 were obtained from regulating gene expression (28). The complex crosstalk Nanjing Chest Hospital Medical School of Southeast Univer- of ceRNA network has been detected in many different sity. Tissues were snap-frozen in RNAlater (Ambion, Austin, diseases (19). Zhang et al (29) analyzed the lncRNA profiles TX, USA) and stored in liquid nitrogen immediately after in 372 hepatocellular cell carcinoma (HCC) patients from surgical resection until total RNA extraction and analysis. The Cancer Genome Atlas (TCGA) (http://cancergenome.nih. Samples were received with a pathology report and a quality gov/) and NCBI GEO Omnibus (GSE65485), and constructed assessment report verifying collection of tumor and/or adja- an lncRNA-miRNA-mRNA network in HCC. Based on the cent non-tumor lung tissues. Informed consent forms were ceRNA hypothesis, Li et al (30) selected 361 RNA sequencing obtained from all patients included in the present study. profiles of gastric cancer (GC) patients from TCGA, and further analysis of the associations between these RNA data RNA sequence data procession and computational analysis. and related patient clinical information. Then, the lncRNA- The LUAD RNA expression data (level 3) of the corresponding miRNA-mRNA ceRNA network of GC was constructed. patients were downloaded from the TCGA Data Portal (March A recent study showed the differential expression of 2016). The RNA sequencing raw reads (lncRNA and mRNA) lncRNA profile of LUAD using high-throughput microarray were post-processed and normalized by TCGA RNASeqV2 analysis from 33 LUAD patients (31). However, studies with system. Level 3, normalized miRNA expression data (the calcu- large scale samples and microarray detection is still rare, and lated expression for all reads aligning to a particular miRNA the relationship between cancer specific lncRNAs and clinical per sample) were downloaded from the TCGA Data Portal features are unclear. TCGA database is a public platform, in performed using Illumina HiSeq and Illumina GA microRNA which sequencing data of lncRNA, miRNA and mRNA for sequencing platforms (Illumina, Inc., Hayward, CA, USA) and LUAD can be downloaded. To improve the reliability and quantile normalized before performing analysis. No further accuracy of the studies, we detected lncRNAs in LUAD using normalizations were applied to the lncRNA, miRNA and data sets from TCGA database. mRNA expression profile data in level 3, because these data In the present study, the RNA sequencing data of 521 LUAD were already normalized by TCGA. To detect the differential samples and 49 adjacent non-tumor lung tissues samples were expression of lncRNA, miRNA and mRNA, samples were collected from TCGA database. This is the first study to inves- divided into LUAD patient tumor tissues vs. adjacent non- tigate the cancer specific lncRNA from large scale sequencing tumor lung tissues, lymph node metastasis of LUAD patients database (TCGA) and ceRNA network constructed in LUAD. vs. non-lymph node metastasis of LUAD patients, well and Quantificational real-time polymerase chain reaction (qRT- moderately-differentiated LUAD (stages I-II) vs. poorly- PCR) was used to verify the bioinformatic analysis results differentiated LUAD (stages III-IV), respectively. For further in LUAD tumor tissues and adjacent non-tumor lung tissues analysis, intersection of lncRNA, miRNA and mRNA was from 53 newly diagnosed LUAD patients. The present study selected. The flow chart for bioinformatics analysis is depicted can help elaborate the function of lncRNAs through lncRNA- in Fig. 1. miRNA-mRNA ceRNA network in LUAD. Functional enrichment analysis. To understand the under- Materials and methods lying biological processes and pathways of aberrantly expressed intersection genes, we use the DAVID (Database Patients and samples. This study was approved by the ethics for Annotation, Visualization, and Integrated Discovery) committee of the Zhongda Hospital Southeast University. Bioinformatics resources (http://david.abcc.ncifcrf.gov/) (32), A total of 521 LUAD cases were obtained from the TCGA to conduct functional enrichment analysis, and we were only database. The exclusion criteria were set as follows: i) histo- interested in GO (Gene Ontology)