Thyroid cancer (TC) is the major endocrine malignancy with estimated 298,000 new cases and 40,000 deaths worldwide in 2012 (1). Thyroid carcinoma is the fastest-growing cancer with the increasing morbidity rate >5% due to the advanced diagnostic techniques as well as multiple risk factors. Although the 5-year survival rate for patients with thyroid carcinoma is relatively higher than other malignant neoplasms, the mortality rate of TC has remained ever-increasing over the past decade (2,3). Thyroid cancer is a heterogeneous disease of 3 histological subtypes: papillary TC (PTC), follicular TC (FTC) and anaplastic TC (ATC) (4). As the most primary malignant thyroid tumor, PTC accounts for up to 80% of total cases. Although PTC is generally effectively treated with thyroidectomy and hormone therapy, large numbers of unresponsive patients suffer from progressive disease and metastases (5). Thus, it is urgent to explore the molecular mechanisms underlying PTC for developing effective diagnostic and therapeutic targets.

Non-coding RNAs (ncRNAs) are generally defined as a wide class of RNAs without protein-coding function, but with universal expression in organisms. ncRNAs are loosely split into small ncRNAs and long non-coding RNAs (lncRNAs), both of which have regulatory functions in the various biological processes (6). The well-documented microRNAs (miRNAs; ~22 nucleotides long) belong to small ncRNAs and are regarded as vital regulators of cellular gene expression network (7). lncRNAs are transcripts >200 nucleotides with tissue-specific expression, but neither has significant open reading frames nor translate into proteins (8). Accumulating studies over the past decades have altered the understanding of lncRNA from ‘dark matter’ to important transcriptional and post-transcriptional regulators. Abnormally expressed lncRNAs participate in tumorigenesis by interrupting biological processes of both oncogenic and tumor suppressive pathways (9). In 2011, Pandolfi et al put forward the competitive endogenous RNA (ceRNA) hypothesis that specific RNAs (including lncRNA, mRNA, pseudogene and circular RNA) can impair miRNA activity by sponging miRNA via common miRNA response elements (MREs) and upregulate target RNA expression subsequently (10). This hypothesis introduced a new RNA-RNA crosstalk theory that can generally regulate gene expression. In addition, the promising area of disclosing the landscape of the ceRNA mechanism has evoked much interest. Therefore, the lncRNA-miRNA-mRNA networks that may lead to tumor development and progression have already been constructed in many malignancies, including gastric cancer (11), hepatocellular carcinoma (12), glioblastoma (13) and lung adenocarcinoma (14).

Recent studies have preliminarily revealed several ceRNA regulatory interactions and corresponding mechanisms in thyroid carcinoma. lncRNA H19 was certified to act as ceRNA by sponging miR-17-5p to upregulate the target gene YES1, demonstrating a potential ceRNA crosstalk including H19, miR-17-5p, and YES1 in thyroid cancer pathogenesis (15). A bioinformatic ceRNA analysis revealed that stem cell factor SOX2 could be functionally co-regulated with other genes by crosstalk mediated by several miRNAs in anaplastic thyroid carcinoma (16). Furthermore, both a ceRNA network built by microarray analysis of 5-paired clinical samples and a protein-coding modulatory network that regulated immune responses have been reported in PTC (17,18). However, a whole genome-wide analysis of the lncRNA-miRNA-mRNA regulatory network of papillary thyroid cancer with large-scale sample size is still lacking.

In the present study, we comprehensively investigated both RNA and miRNA sequencing data of 348 PTC primary tumor tissues and 58 non-cancerous thyroid samples from The Cancer Genome Atlas (TCGA) data matrix to obtain the aberrantly expressed lncRNAs, miRNAs, and mRNAs. Next, an lncRNA-associated ceRNA network of papillary thyroid cancer was built based on ceRNA theory. The expression levels of the important lncRNAs involved in ceRNA crosstalk were also assessed for their prognostic values in patients with papillary thyroid cancer.

PTC is the most frequent endocrine neoplasia characterized by a variety of aberrant molecular events driving tumori-genesis (26). Currently, much effort has been made to elucidate the roles of ncRNAs involved in tumorigenesis, disease progression and metastasis of PTC (17,27,28). According to the ceRNA hypothesis, increasing evidence has demonstrated that lncRNAs regulated target genes through microRNAs (miRNAs) competition (29). To comprehensively identify the landscape regarding how lncRNA-associated ceRNA network affects PTC, the present study analyzed the large-scale sequencing data of PTC patient cohort in TCGA database. We identified the aberrantly expressed lncRNAs, mRNAs and miRNAs, and further successfully constructed the dysregulated lncRNA-associated ceRNA network in PTC by biological prediction. We also identified several lncRNAs and their related mRNAs to be potential prognostic indicators for PTC patients.

In previous studies, several groups have identified numerous abnormally expressed lncRNAs in PTC compared with non-tumor tissues. Two small cohort studies based on microarray sequencing of 3 and 5 pairs of PTC samples, respectively, have identified thousands of significantly dysregulated lncRNAs and validated several DElncRNAs by real-time PCR (18,30). Xie et al reported a ceRNA network in PTC containing 29 lncRNAs, 9 miRNAs and 67 mRNAs by microarray analysis of 5-paired clinical samples (31). However, lncRNA-associated ceRNA network based on whole-genome gene expression profiling with large study population in PTC has not been described.

In the present study, 780 lncRNAs were detected as commonly dysregulated lncRNAs in 3 PTC variants (CPTC, FPTC and TCPTC) compared with normal tissues, including 440 upregulated and 340 downregulated. Among them, 45 aberrantly expressed lncRNAs were identified in the ceRNA network, and 8 out of them were related to tumor progression. The low expression of an intergenic lncRNA LINC00473 in PTC tumor tissues (~10-folds) may have potential capacity to promote tumor growth and pathologic stage. It has been identified to have prognostic value for lung cancer in a cyclic AMP-dependent manner (32). However, whether LINC00473 can utilize similar mechanism to lung cancer in PTC needs further research. LINC00460, a human gene transcribed from chromosome 13, is upregulated in various cancers, including thyroid cancer (33). The upregulation of LINC00460 was also observed in the present study and was positively correlated with the pathologic stage of PTC. It was reported that LINC00460 participated in a series of biological processes (e.g., metabolic process, chromatin modification, cell cycle and cell death) and showed prognostic prediction power for head and neck squamous cell carcinoma (33,34). These findings implied that LINC00460 may functionally be involved in the tumorigenesis of PTC. Moreover, RBMS3-AS1 and ZFX-AS1 also had potential values for PTC patients prognosis. Since these lncRNAs are reported for the first time as prognostic predictors, much effort deserves to be made to elucidate their biological function in neoplasms. Although the lncRNAs listed in Table V had no statistical significance in Kaplan Meier analysis (data not shown), both the lnRNAs that were associated with the progression of PTC and with patients' overall survival may have potential roles in PTC and are worth studying in the future.

However, having identified several specific lncRNAs for PTC, the present study drew a comprehensive ceRNA network, which revealed the complexity of the regulatory relationships among lncRNAs, miRNAs and mRNAs. E2F1, the best-studied E2F member, was significantly upregulated in PTC tumor tissues, which was consistent with a previous study (35). E2F1 was involved in both cancer-related pathways: pathways in cancer and miRNAs in cancer. Although previously E2F1 was found to promote thyroid carcinogenesis (35), our results demonstrated that high expression of E2F1 could prolong survival time of PTC patients, which strongly suggested its tumor-suppressive activity in thyroid cancer. Thus, we supposed that E2F1 may exhibit both oncogenic and tumor suppressive properties in thyroid cancer resulting from its capacity to induce tumor cell proliferation and apoptosis (36–38). The ceRNA network predicted that E2F1 was a target gene of miR-205. It was consistent with the previous study that miR-205 induced cancer senescence by regulating E2F1 and ultimately suppressed melanoma development (39). Our bioinformatic analysis predicted that miR-205 may interact with LINC00284, the increased expression of which was also positively correlated with the prognosis of PTC patients. Although the function of LINC00284 is poorly annotated, it is conceivable that LINC00284 may act as a ceRNA to interact with miR-205 and E2F1 in PTC.

There are several limitations in the present study. First, ceRNA hypothesis indicates that lncRNAs can serve as ceRNAs to regulate expression of the target genes by merging miRNAs. Therefore, the complex ceRNA network of PTC was built based on this hypothesis using biological prediction. Second, the ceRNA network with hundreds of connections suggested that one lncRNA connected with a number of miRNAs and relevant mRNAs. In this regard, our further study may focus on conducting experiments to validate several crucial interactions, particularly to verify the key interactions we discussed above and determine the roles of these lncRNAs as ceRNA in PTC. Third, the 3 lncRNAs that were aberrantly expressed in race need extensive validation studies, which must be based on a large-scale multiracial cohort.

In conclusion, we identified various dysregulated lncRNAs, miRNAs and mRNAs between tumor and non-tumor samples by genome-wide analysis of a large PTC patient cohort from TCGA. The lncRNA-related ceRNA network will be useful to guide further investigation concerning the involved lncRNAs of their definite mechanisms and functions in the tumorigenesis of PTC.