Tahira et al. Molecular Cancer 2011, 10:141 http://www.molecular-cancer.com/content/10/1/141 RESEARCH Open Access Long noncoding intronic RNAs are differentially expressed in primary and metastatic pancreatic cancer Ana C Tahira1, Márcia S Kubrusly2, Michele F Faria1, Bianca Dazzani1, Rogério S Fonseca1, Vinicius Maracaja-Coutinho1, Sergio Verjovski-Almeida1, Marcel CC Machado2 and Eduardo M Reis1* Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is known by its aggressiveness and lack of effective therapeutic options. Thus, improvement in current knowledge of molecular changes associated with pancreatic cancer is urgently needed to explore novel venues of diagnostics and treatment of this dismal disease. While there is mounting evidence that long noncoding RNAs (lncRNAs) transcribed from intronic and intergenic regions of the human genome may play different roles in the regulation of gene expression in normal and cancer cells, their expression pattern and biological relevance in pancreatic cancer is currently unknown. In the present work we investigated the relative abundance of a collection of lncRNAs in patients’ pancreatic tissue samples aiming at identifying gene expression profiles correlated to pancreatic cancer and metastasis. Methods: Custom 3,355-element spotted cDNA microarray interrogating protein-coding genes and putative lncRNA were used to obtain expression profiles from 38 clinical samples of tumor and non-tumor pancreatic tissues. Bioinformatics analyses were performed to characterize structure and conservation of lncRNAs expressed in pancreatic tissues, as well as to identify expression signatures correlated to tissue histology. Strand-specific reverse transcription followed by PCR and qRT-PCR were employed to determine strandedness of lncRNAs and to validate microarray results, respectively. Results: We show that subsets of intronic/intergenic lncRNAs are expressed across tumor and non-tumor pancreatic tissue samples. Enrichment of promoter-associated chromatin marks and over-representation of conserved DNA elements and stable secondary structure predictions suggest that these transcripts are generated from independent transcriptional units and that at least a fraction is under evolutionary selection, and thus potentially functional. Statistically significant expression signatures comprising protein-coding mRNAs and lncRNAs that correlate to PDAC or to pancreatic cancer metastasis were identified. Interestingly, loci harboring intronic lncRNAs differentially expressed in PDAC metastases were enriched in genes associated to the MAPK pathway. Orientation-specific RT- PCR documented that intronic transcripts are expressed in sense, antisense or both orientations relative to protein- coding mRNAs. Differential expression of a subset of intronic lncRNAs (PPP3CB, MAP3K14 and DAPK1 loci)in metastatic samples was confirmed by Real-Time PCR. Conclusion: Our findings reveal sets of intronic lncRNAs expressed in pancreatic tissues whose abundance is correlated to PDAC or metastasis, thus pointing to the potential relevance of this class of transcripts in biological processes related to malignant transformation and metastasis in pancreatic cancer. Keywords: pancreatic cancer, molecular markers, noncoding RNAs, intronic transcription, metastasis, MAPK , path- way, cDNA microarrays * Correspondence: [email protected] 1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900, São Paulo, SP, Brasil Full list of author information is available at the end of the article © 2011 Tahira et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tahira et al. Molecular Cancer 2011, 10:141 Page 2 of 19 http://www.molecular-cancer.com/content/10/1/141 Background targets [14,16-19]. Increased expression of the lncRNA Pancreatic ductal adenocarcinoma (PDAC) is the most MALAT-1 has been observed in several types of tumors, common pancreatic neoplasm and accounts for > 85% including metastatic non-small cell lung cancer [19]. of pancreatic tumor cases [1]. PDAC is a devastating Recently, augmented levels of HOTAIR in primary disease with very poor prognosis for which the only breast tumors were shown to correlate with breast can- curative treatment is resection surgery [2]. However, cer invasiveness and metastasis [18]. Measurement of only 15-20% of patients have resectable pancreatic lncRNA PCA3 in patient urine samples has been shown tumor, and from these only 20% presents a 5-year survi- to allow more sensitive and specific diagnosis of prostate val, which results in an average 5-year survival rate of 3- cancer than the widely used marker prostate-specific 5% [1]. PDAC aggressiveness is mainly associated to the antigen (PSA) [16]. The lncRNA HULC is highly lack of early diagnosis tools and the limited response to expressed in hepatocarcinoma patients and detected in available treatments [2]. the blood by conventional PCR methods [20]. Large-scale gene expression studies of tumor samples There are several reports of aberrant expression of have been extensively employed to delineate the mole- microRNAs in PDAC [21,22], and there is potential in cular pathways and cellular processes involved in tumor- their use as biomarkers for disease diagnosis [23,24]. igenesis and progression of PDAC [3] and to search for However, there is a paucity of information regarding the novel biomarkers for diagnosis and molecular targets for expression of lncRNAs in pancreatic cancer. In an inter- therapeutic intervention in pancreatic cancer [4]. In esting study performed by Ting et al. it was observed spite of the wealth of information generated in recent the aberrant overexpression of satellite repeat RNAs years on the most frequent molecular alterations found (HSATII) ranging from 100 to 5000 nt in patients with in PDAC [5], there are still important open question in PDAC [25]. Interestingly, detection of HSATII by RNA pancreatic cancer biology such as the profound resis- in situ hybridization was able to correctly diagnose tance of primary and metastatic PDAC to chemo- and PDAC in tumor biopsies, including cases in which the radiotherapy [6]. Regarding the identification of molecu- histopathology was non-diagnostic [25]. lar markers for pancreatic cancer diagnostic/prognostic, Our group has previously shown that most (at least while some promising candidate genes have been pro- 74%) annotated protein-coding gene loci generate intra- posed [4], none have been proven effective to signifi- genic lncRNAs that map to intronic regions [26]. Possi- cantly improve early detection and to reduce mortality/ ble relevance of intronic lncRNAs to neoplastic morbidity of the disease. Thus, a better understanding processes was proposed following the observation that of the molecular basis of pancreatic cancer is required subsets of these transcripts are present in gene expres- for the identification of more effective diagnostic mar- sion signatures correlated to the degree of malignancy kers and therapeutic targets. in prostate cancer [17] or to tissue histology in head Over the last decade, advances in genome-wide ana- and neck tumors [27] and renal cell carcinoma [28]. In lyses of the eukaryotic transcriptome have revealed addition, a number of intronic lncRNAs were found to that the majority of the human genome is transcribed, be regulated by androgen stimulation of cultured pros- producing large numbers of long (> 200 nt) noncoding tate cancer cells [29], indicating that these transcripts RNAs (lncRNAs) mapping to intronic and intergenic are expressed in a regulated manner and thus, corrobor- regions [7-10]. These include subsets of polyadenylated ating the idea that intronic lncRNAs are biologically and non-adenylated transcripts that accumulate differ- relevant. ently in the nucleus and cytoplasm of cells [10,11]. In this study, we used a custom cDNA microarray While only a small fraction of lncRNAs have been platform with probes for lncRNAs expressed from intro- characterized in detail, it is clear that these transcripts nic and intergenic regions of the human genome, as may act through diverse molecular mechanisms and well as for a selected set of cancer-related protein-cod- play regulatory and structural roles in important biolo- ing genes to generate expression profiles from a collec- gical processes, such as in genomic imprinting, chro- tion of tumor and non-tumor pancreatic tissue samples. mosome inactivation, cell differentiation and Expression of intronic/intergenic lncRNAs subsets was development, cell proliferation, protein nuclear import, detected across all samples tested. Enrichment of pro- organization of nuclear domains and apoptosis (see moter-associated chromatin marks indicate that these [12] for a review). transcripts originate from independent transcriptional Altered expression of lncRNAs has been documented units. Over-representation of conserved DNA elements in different types of human cancer [13-15] prompting and stable secondary structure predictions suggest that an increasing interest in their use as biomarkers for at least a fraction of these transcripts are under evolu- diagnosis and prognosis as well as potential therapeutic tionary selection and thus potentially functional. Tahira et al. Molecular Cancer 2011, 10:141 Page 3 of 19 http://www.molecular-cancer.com/content/10/1/141