MicroRNA-dependent regulation of transcription in non-small cell lung cancer

MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer Sonia Molina-Pinelo 1 , Gabriel Gutie ´rrez 2 , Maria Dolores Pastor 1 , Marta Hergueta 3,13 , Gema Moreno- Bueno 3,13 , Rocı ´o Garcı ´a-Carbonero 1,4 , Ana Nogal 1,5 , Rocı ´o Sua ´rez 1 , Ana Salinas 1 , Francisco Pozo- Rodrı ´guez 6,7 , Fernando Lopez-Rios 8 , Maria Teresa Agullo ´-Ortun ˜o 9 , Irene Ferrer 1 , Asuncio ´n Perpin ˜a ´ 10 , Jose ´Palacios 11 , Amancio Carnero 12 , Luis Paz-Ares 1,4 * 1Molecular Oncology and New Therapies Group. Instituto de Biomedicina de Sevilla (IBIS) (HUVR, CSIC, Universidad de Sevilla), Sevilla, Spain, 2Genetic Department, Universidad de Sevilla, Seville, Spain, 3Biochemistry Department, Universidad Auto ´noma de Madrid, Instituto de Investigaciones Biome ´dicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain, 4Medical Oncology Department, Hospital Universitario Virgen del Rocio, Sevilla, Spain, 5Instituto de Cie ˆncias Biome ´dicas Abel Salazar (ICBAS), Porto, Portugal, 6Service of Neumology, Hospital 12 de Octubre, Madrid, Spain, 7Centro de Investigacio ´n Biome ´dica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain, 8Pathology Department, Laboratorio de Dianas Terape ´uticas, Centro Integral Oncolo ´gico Clara Campal, Madrid, Spain, 9Service of Oncology, Instituto de Investigacio ´n Sanitaria i+12, Hospital Doce de Octubre, Madrid, Spain, 10 Service of Neumology, Hospital Severo Ochoa, Madrid, Spain, 11 Department of Pathology, Hospital Universitario Ramo ´n y Cajal, Instituto de Investigacio ´n Sanitaria Ramo ´n y Cajal (IRYCIS), Madrid, Spain, 12 Molecular Biology of Cancer Group, Instituto de Biomedicina de Sevilla (IBIS)/(HUVR, CSIC, Universidad de Sevilla), Seville, Spain, 13 IdiPAZ (Instituto de Investigacio ´n Sanitaria La Paz) & Fundacio ´n MD Anderson International, Madrid Spain Abstract Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR- 422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies. Citation: Molina-Pinelo S, Gutie ´rrez G, Pastor MD, Hergueta M, Moreno-Bueno G, et al. (2014) MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer. PLoS ONE 9(3): e90524. doi:10.1371/journal.pone.0090524 Editor: Bernard Mari, IPMC, CNRS UMR 7275 UNS, France Received August 29, 2013; Accepted February 3, 2014; Published March 13, 2014 Copyright: ß2014 Molina-Pinelo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: LPA is funded by Fondo de Investigacio ´n Sanitaria (PI081156, PI1102688 and R12/0036/0028), Proyecto de Excelencia de la Consejerı ´a de Innovacio ´n, Ciencia y Empresa, Junta de Andalucı ´a (P08-CVI-04090), and the Roche Fellowship in 75th Anniversary, Spain. SMP is funded by Fondo de Investigacio ´n Sanitaria (CD1100153), Fundacio ´n Cientı ´fica de la Asociacio ´n Espan ˜ola Contra el Ca ´ncer, Consejerı ´a de Salud, Servicio Andaluz de Salud (PI-0224/2009 and PI-0046/2012), and Fundacio ´n Mutua Madrilen ˜a (2009). MDP is funded by Fondo de Investigacio ´n Sanitaria (CD0900148). RGC is funded by Fondo de Investigacio ´n Sanitaria (PI10/02164). GMB is funded by SAF2010-20175 and Madrid Regional Government (S2010/BMD-2303). AC lab was supported by grants to from the Spanish Ministry of Economy and Competitivity, ISCIII (PI12/00137, RTICC: RD12/0036/0028), Consejeria de Ciencia e Innovacion (CTS-6844) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Lung cancer is the primary cause of cancer death worldwide, being responsible for one million deaths annually [1]. Non-small cell lung cancer (NSCLC) accounts for 80% of all lung tumours, and includes several histological subtypes such as large cell carcinoma (LCC), squamous cell carcinoma (SCC), and adenocarcinoma. SCC and adenocarcinoma are the most common types of NSCLC, accounting for 25% and 40% of all cases, respectively [2,3]. SCC derives from dysplastic multilayer epithelium in the central airways, whereas adenocarcinoma originates preferentially from precursor cells of the mono- or bilayer surface epithelium of the lung periphery [4]. NSCLC, regardless of the histological subtype, has been traditionally treated in the clinic as a single homogeneous entity. However, increasing evidence illustrates the great biological diversity of this disease, which is progressively leading to more specific diagnostic and therapeutic strategies depending upon the histological subtype concerned. Indeed, advances in targeted lung cancer therapy now PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e90524 demand accurate classification of NSCLC [5]. For example, EGFR mutations are more prevalent in patients with lung adenocarcinoma, and the presence of these mutations is associated with sensitivity to EGFR tyrosine kinase inhibitors [6]. Similarly, ALK traslocations, present in only 4% of adenocarcinomas, are predictive of a high sensitivity to ALK-directed therapies such as crizotinib. By contrast, FGFR1 amplification is more commonly observed in SCC, and is now being considered a potentially actionable target in clinical trials with FGFR inhibitors [7]. Therefore, a greater knowledge of the molecular mechanisms involved in the genesis, progression and spread of the different subtypes of NSCLC is necessary for the development of specific diagnostic methods and the design of more adequate, individualized and effective therapeutic strategies. The great advances in genomic technologies have generated many candidate biomarkers with potential clinical value in NSCLC. MicroRNAs, as post-transcriptional modulators, are key players in the regulation of many biological processes. Dysregulation of their physiological roles contributes to many pathological conditions, including the initiation and progression of cancer. In this context, a number of studies have assessed the potential role of miRNA signatures to discriminate histological subtypes or to predict recurrence or survival of NSCLC patients [8,9,10,11,12,13,14], and miRNA profiling has been proposed as a highly reliable strategy for classifying NSCLCs [11,15,16]. Nevertheless, the high complexity of transcriptome regulation complicates the full understanding of gene regulatory networks involved in these processes. To address this issue, the aim of this study was to assess miRNA- dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. With this purpose, miRNA and mRNA paired expression profiles were analyzed in NSCLC tumor samples, and the potential interactions among them were explored. In this study we have identified and validated a subset of deregulated miRNAs and target genes that are able to define distinct molecular features of these two major histological subtypes of NSCLC. Materials and Methods Patients and Tumor Specimens Patients included in this study were required to have histologically confirmed early stage SCC or adenocarcinoma NSCLC. Tumor samples from 85 patients were prospectively collected during the surgical procedure and immediately snapfrozen at 280uC until further use. Adjacent non-tumor lung tissue was also collected from patients included in the validation cohort. The study protocol was approved by the institutional review boards of participating centers [Hospital Universitario Doce de Octubre (Madrid) and Hospital Universitario Virgen del Rocı ´o (Sevilla)] and all patients provided written informed consent prior to study entry. Clinical and pathologic data were extracted from the medical records and centrally reviewed for the purpose of this study. The study population was divided in a training cohort (N = 44) that was used for profile development and an independent validation cohort (N = 41). Main characteristics of study population are summarized in Tables 1 and 2. Microarray Gene Expression Profiles Microarray experiments were performed using Human Whole Genome 44 K array G4112A (Agilent technologies, Wilmington, DE). RNA was isolated using Trizol (Invitrogen) and RNAesy Extraction Kit (QIAGen, Germany) as indicated by the manufacturers. RNA was labeled and array hybridized using the Low RNA Linear Amplification Kit and the In Situ Hybridization Kit Plus (Agilent technologies, Wilmington, DE) respectively. After hybridization and washing, the slides were scanned in an Axon GenePix Scanner (Axon Instruments Inc., Union City, CA) and analyzed using Feature Extraction Software 6.1.1 (Agilent technologies, Wilmington, DE). RNA from tumor samples were labeled with Cy5-dUTP, and hybridized against a lung cancer reference pool (labeled with with Cy3-dUTP) consisting of primary tumor tissue from patients with different histological subtypes of lung cancer. As control, ten additional hybridizations were performed using the reciprocal fluorochrome labeling. To detect differentially expressed genes between the two histological subtypes, two types of analysis were undertaken with the MIDAW tool [17]. First, a t-test was performed with false discovery rate (FDR) control estimated using the single-step Bonferroni procedure. Genes that passed the t-test filter were subjected to a second filter. Only genes showing a mean log ratio value lower that 20.3 or greater than 0.3 (equivalent to a 2-fold change) were selected as differentially expressed. Second, a discriminant analysis for the identification of the set of best marker genes was performed based on the Prediction Analysis of Microarray (PAM) algorithm. Microarray raw data tables have been deposited in the Gene Expression Omnibus under the accession number of GSE42998. MicroRNA qRT-PCR Assay Total RNA, containing small RNA, was extracted from tumour tissue samples by mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The total RNA yield was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Tech, DE, USA). The Agilent 2100 Bioanalyzer was used to determine the quantity and quality of the RNA samples (Agilent, Palo Alto, CA). Mature human miRNA expression was detected and quantified using the TaqMan Low Density Arrays (TLDA) based on Applied Biosystems’ 7900 HT Micro Fluidic Cards (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. The Human MicroRNA Card Set v2.0 array (Catalog Number 4400238) is a two card set containing a total of 384 TaqMan MicroRNA Assays per card to enable accurate quantification of 667 human microRNAs, all catalogued in the miRBase database. TLDAs were performed in a two-step process. Briefly, during the first step, 450 ng of total RNA were reverse transcribed using Megaplex RT Primers and the TaqMan miRNA reverse transcription kit in a total volume of 7.5 ml. The 7.5 ml reactions were incubated in a G-Storm Thermal Cycler (Gene Technologies, Essex, UK) for 2 min at 16uC, 1 min at 42uC, and 1 min at 50uC during 40 cycles, held for 5 min at 85uC, and then kept at 4uC. In the second step, 6 mL of cDNA sample and TaqMan Universal PCR master mix were loaded in fill ports on the TLDA microfluidic card. The card was briefly centrifuged for 1 min at 331 g to distribute samples in the multiple wells connected to the fill ports and then sealed to prevent well-to-well contamination. The reactions were incubated in a 384 well plate at 50uC for 2 sec and 94.5uC for 10 min, followed by 40 cycles of 30 sec at 97uC and 1 min at 59uC. Finally, the cards were processed and analyzed on an ABIPrism 7900 HT Sequence Detection System. TLDA raw data tables have been deposited in the Gene Expression Omnibus under the accession number of GSE43000. Expression of target miRNAs was normalized to the expression of RNU48. One non-human miRNA, was used in each experiment as a negative control. Cycle threshold (Ct) values were calculated using the SDS software v.2.3 using automatic baseline settings and a threshold of 0.2. Relative quantitation of miRNA expression was calculated by the 2 2DCt method (Applied Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 2 March 2014 | Volume 9 | Issue 3 | e90524 Biosystems user bulletin no. 2 (P/N 4303859)). Only miRNA detectable in at least 80% of samples were considered for evaluation. Significance of miRNA expression differences observed between the two histolofical subgroups (adenocarcinoma and SCC) was assessed by the t-test. mRNA and miRNA Expression Correlation Assessment To evaluate the potential association between differentially expressed mRNA and miRNA observed in our study, we searched for the transcriptional targets of the identified miRNAs in three web databases for miRNA target prediction: miRanda [18], TargetScan release 6.0 [19], and miRWalk [20]. Putative target genes that matched with those found to be disregulated in our patient population were selected for further validation by qPCR. Validation of Microarray Gene Expression Profiles by qPCR Eleven differentially expressed genes among the two study conditions (SCC and adenocarcinoma NSCLC), identified as putative targets of several dis-regulated miRNAs, were selected for further validation by qPCR in the original training cohort and then in an independent validation cohort. The RNA was reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Briefly, single-stranded cDNA was synthesized from 1 mg total RNA in 10 mL reaction volume, according to the manufacturer’s protocol. The reaction was incubated at 25uC for 10 min followed by 120 min at 37uC and inactivation at 85uC for 5 min. The TaqMan Gene Expression Assay system (Applied Biosystems) was used for quantitating transcription levels of selected genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1, DMRT2, DSC3 and KRT6A). Three endogenous control genes (B2M,ACTB and GAPDH) and one no-template-control (NTC) were also run for each RNA sample. We chose B2M for normalization across different genes as this gene showed the most relatively constant expression across different tissue samples (data not shown). The gene expression for each gene was determined using the median expression level of the three technical replicates. PCR reactions were performed on an Applied Biosystems 7900HT Sequence Detection system in 10 mL volumes at 95uC for 10 min, followed by 40 cycles of 95uC for 15 sec and 60uC for 1 min. Ct values were obtained with the SDS software v.2.3 (Applied Biosystems). Relative quantification of mRNA expression was calculated by the 2 2DCt method (Applied Biosystems user bulletin no. 2 (P/N 4303859)). Validation of miRNA TLDA Expression Profiles by qPCR Expression of nine selected miRNAs (miR-149, miR-205, miR- 375, miR-378, miR-422a, miR-483-5p, miR-494, miR-601 and miR-708) was assessed in the independent validation cohort by the specific TaqMan MicroRNA assays according to the manufacturer’s instructions (Applied Biosystems). Briefly, 2 ng/mL of total Table 1. Summary of patients’ characteristics of the training cohort. Variable Subvariable Squamous cell carcinoma (N = 25) Adenocarcinoma (N = 19) Age years 71 [62–74] 65 [59–74] Gender Male 25 (100%) 17 (89.4%) Female – 2 (10.6%) Stage I–IIB 17 (68%) 16 (84.2%) IIIA 7 (28%) 2 (10.5%) IIIB–IV 1 (4%) 1 (5.3%) Smoking Status Current smoker 9 (36%) 7 (36.8%) Ex-smoker 16 (64%) 10 (52.7%) Never smoker – 2 (10.5%) Continuous variables are expressed as median [interquartile range (IQR)] and categorical ones as number of cases (%). doi:10.1371/journal.pone.0090524.t001 Table 2. Summary of patients’ characteristics of the validation cohort. Variable Subvariable Squamous cell carcinoma (N = 25) Adenocarcinoma (N = 19) Age years 66 [60–74] 66 [63–73] Gender Male 15 (88.2%) 16 (66.7%) Female 2 (11.8%) 8 (33.3%) Stage I–IIB 15 (88.2%) 21 (87.5%) IIIA 2 (11.7%) 2 (8.3%) IIIB–IV – 1 (4.2%) Smoking Status Current smoker 9 (52.9%) 6 (25.0%) Ex-smoker 8 (47.1%) 15 (62.5%) Never smoker – 3 (12.5%) Continuous variables are expressed as median [interquartile range (IQR)] and categorical ones as number of cases (%). doi:10.1371/journal.pone.0090524.t002 Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 3 March 2014 | Volume 9 | Issue 3 | e90524 RNA was converted into cDNA by reverse transcriptase reaction that was performed by sequential incubation at 16uC for 30 min, 42uC for 30 min and 85uC for 5 min. PCR reaction mixture (10 mL) contained 0.66 mL of RT product, 5 mL of TaqMan 2X Universal PCR Master Mix and 0.5 mL of the appropriate TaqMan MicroRNA Assay (20X) containing primers and probe for the miRNA of interest (Applied Biosystems). The mixture was initially incubated at 95uC for 10 min, followed by 40 cycles of 95uC for 15 seconds and 60uC for 60 seconds. MicroRNA expression was quantified by the comparative 2 2DDCt method, normalizing Ct values to RNU48. In the validation cohort, tumor expression values were additionally normalized to expression values in paired adjacent normal lung tissue. 39-UTR Reporter Assay for miR Target Validation Confirmation of miR-149-binding to the 39UTR of ABCC3 and of miR-378 and miR-422-binding to the 39UTR of TMEM45B. HEK 293 cells at 80% confluency were cotransfected with luciferase reporter plasmids harboring the complete 39-UTR of the desired gene (SwitchGear Genomics) along with 100 nM of each miR-mimic or miRNA control (Sigma). DharmaFECT Duo (Thermo Scientific) was used as the transfection reagent in Opti-MEM (Life Technologies). Luminescence was assayed 24 hours later using LightSwitch Assay Reagents (SwitchGear Genomics) according to the manufacturer’s instructions. Knockdown was assessed by calculating luciferase signal ratios for specific miRNA/non-targeting control, using empty reporter vector as control for non-specific effects. Each experiment was performed in triplicate. t -test was performed for wells from multiple experiments, and we compared mimictransfected cells with a mimic control for each gene vector. Diagnostic Performance Assessment of Selected Genes Diagnostic performance parameters were calculated for selected genes in 2x2-contingency tables. Confidence intervals for these parameters were calculated with the Pearson method based on the F distribution. As sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) are statistical measures of the performance of a binary classification test, gene expression values were converted to binary variables with the median expression value as the reference value (high versus low expression). These parameters were calculated for each validated miRNA/target mRNA pair. The concurrent occurence of high miRNA expression and low target mRNA expression in the appropriate histological condition (SCC or adenocarcinoma) was considered a true positive test. Sensitivity or true positive rate measures the proportion of actual positives which are correctly identified. Specificity or true negative rate measures the proportion of negatives which are correctly identified. The PPV describes the probability of having the condition given a positive screening test result in the analyzed population. The NPV describes the probability of not having the condition given a negative screening test result in the analyzed population. Results Profile Development Gene expression profiles by histological subtype. Whole genome expression arrays were performed in tumor samples of patients of the training cohort, and expression profiles of SCC and adenocarcinoma tumor types were compared one gene at a time using the one-sample t-test. After single-step Bonferroni adjustment, 727 genes were identified to be differentially expressed by more than two fold in either histological subtype relative to the reference pool (Tables A, B, C and D in Table S1). Of these 727 genes, five were up-regulated and 195 down-regulated in patients with adenocarcinoma, and 13 were up-regulated and 516 downregulated in patients with SCC. Additionally, a second independent evaluation of mRNA differential expression was performed by discriminant microarray data analysis to minimize false-positive findings. The Prediction Analysis of Microarray (PAM) algorithm identified 61 genes that defined a molecular signature able to discriminate adenocarcinoma from SCC samples (figure 1). Of these 61 genes, 56 matched deregulated genes found by the previously performed one-sample t-test, and were therefore selected for further analysis and validation. MicroRNA expression profile by histological subtype. MicroRNAs TLDA arrays were performed in tumor samples of patients of the training cohort. Nine miRNAs (miR- 149, miR-205, miR-375, miR-378, miR-422a, miR-483-5p, miR- 494, miR-601 and miR-708) were found to be differentially expressed between the SCC and adenocarcinoma histological subtypes by a FDR-corrected threshold of 0.05. Eight of these 9 miRNAs were over-expressed in SCC compared to adenocarcinoma, and one (miR-375) was over-expressed in adenocarcinoma compared to SCC (Table 3). MicroRNA target prediction. Eleven of the 56 genes (20%) found to be deregulated by tumor type in our study were found to be putative targets of at least one of the 9 miRNAs also identified to be differentially expressed in our study population according to histological subtype (SCC versus adenocarcinoma). For the 8 overexpressed miRNAs in SCC, 8 mRNA (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B and MUC1) were predicted as targets by several algorithms. These genes were found to be downregulated in SCC compared to adenocarcinoma in our study (Table 2). Three of these 8 genes (CEACAM6, MLPH and TMEM45B) were predicted targets of more than one of these miRNAs (figure 2). As shown in table 2, three genes (DSC3, KRT6A and DMRT2) were predicted as targets of miR-375, the miRNA upregulated in adenocarcinoma, and these genes were consistently down-regulated in this histological subtype. Profile Validation Validation of gene differential expression by quantitative RT-PCR. Eleven deregulated genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1, DSC3, DMRT2 and KRT6A), identified as putative target genes of deregulated miRNAs in our study, were then selected for further validation by rtPCR both in the training cohort and in an independent cohort of patients. In the training cohort, 9 of the 11 genes tested were confirmed to be differentially expressed by histological subtype by quantitative PCR (figure 3). CEACAM5,CLDN3, CGN, ABCC3, MUC1, ACSL5,MLPH and TMEM45B were significantly down-regulated in SCC, and KRT6A was significantly down-regulated in adenocarcinoma. Based on results obtained in the training cohort, 9 mRNA and 9 miRNAs were selected for further validation by Taqman-based RT-qPCR in tumor and matched normal tissue from an independent cohort of lung cancer patients. In this cohort, expression patterns of mRNAs were consistent with those quantified in the training cohort. Expression patterns by histological subtype of all 9 mRNAs tested resembled those observed in the training cohort, and differences observed among subgroups were all statistically significant (figure 4). Regarding the 9 miRNAs, five (miR-149, miR-205, miR-378, miR-422a and miR-708) were Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 4 March 2014 | Volume 9 | Issue 3 | e90524 Figure 1. Distinct transcriptional profiles of SCC and adenocarcinoma histological subtypes. The Prediction Analysis of Microarray algorithm identified 61 genes that defined a molecular signature for each histological subtype. The modulators’ dendrogram represents an unsupervised hierarchical clustering analysis of 19 adenocarcinoma and 25 SCC based on their gene expression profile. The heat map was color coded using red for up-regulation and green for down-regulation from a lung cancer reference pool. On the top of the heap map, colours correspond to gene expression profiles of SCC samples (blue) versus adenocarcinoma samples (red). doi:10.1371/journal.pone.0090524.g001 Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 5 March 2014 | Volume 9 | Issue 3 | e90524 Table 3. Identification of down-regulated mRNA from deregulated miRNAs in SCC and adenocarcinoma. Target genes predicted Prediction programs Over-expressed miRNAs in SCC miR-149 ABCC3 miRanda, miRWalk, TargetScan MUC1 TargetScan CEACAM6 miRanda, miRWalk, TargetScan CGN miRanda CLDN3 miRanda miR-205 ACSL5 miRanda, TargetScan MLPH miRWalk, TargetScan CEACAM6 miRanda, miRWalk, TargetScan miR-378 TMEM45B miRanda miR-422a TMEM45B miRanda miR-483-5p TMEM45B miRanda, TargetScan miR-494 ACSL5 TargetScan MLPH miRanda CEACAM6 miRanda miR-601 MLPH miRanda, miRWalk, TargetScan miR-708 CEACAM6 miRanda, TargetScan Over-expressed miRNAs in adenocarcinoma miR-375 DSC3 miRanda, miRWalk, TargetScan KRT6A miRanda DMRT2 miRanda, miRWalk doi:10.1371/journal.pone.0090524.t003 Figure 2. MicroRNA-mRNA target networks. The graph shows the transcriptional targets of differently expressed miRNAs in SCC and adenocarcinoma tumor types using three web databases of miRNA target prediction (miRANDA, TargetScan and miRWalk). The default colour scheme used to represent expression level is red/blue (red for over-expression of mRNAs or miRNAs in SCC versus adenocarcinoma and blue for down-expression of mRNAs or miRNAs in SCC versus adenocarcinoma). The arrows indicate mRNA repression by the connected miRNAs. Squares represent deregulated mRNAs and ovals represent differentially expressed miRNAs in lung adenocarcinoma and SCC. doi:10.1371/journal.pone.0090524.g002 Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 6 March 2014 | Volume 9 | Issue 3 | e90524 found to be significantly over-expressed in SCC and miR-375 was significantly over-expressed in adenocarcinoma (figure 5). Correlation between miRNA and mRNA expression. To study the functional relevance of miRNAs in the regulation of specific mRNAs identified as potential biomarkers, we analyzed in the validation cohort the correlation between miRNAs and predicted target-mRNAs expression in each patient (figure 6). An inverse correlation was observed between ABCC3, MUC1 and CEACAM6 and miR-149 expression levels. Moreover, higher levels of CEACAM6 were associated with lower levels of miR-205 and miR-708, being the correlation significant for miR-708 (r = 2 0362; p = 0.030). ACSL5 and KRT6A had a statistically significant correlation with miR-205 (r = 20.475; p = 0.003) and miR-375 (r = 20.311; p = 0.065), respectively. In the case of TMEM45B, significant correlations were found for miR-378 and miR-422a (r = 2 0.394, p = 0.016 and r = 20.413, p = 0.015, respectively). These results suggest a potential role of miRNAs in the regulation of these genes. Subsequently, some of these targets were tested using luciferase reporter gene assays. We achieved that overexpression of miR-149 in HEK 293 cells down regulates the luciferase activity of reporter construct containing the ABCC3 3-UTR (figure 7). This shows that miR-149 binds directly to this target RNA and inhibits their expression. In addition, overexpression of miR-378 and miR- 422a significantly inhibit TMEM45B expression (figure 7). Diagnostic Performance of Selected Genes to Discriminate SCC from Adenocarcinoma Histological NSCLC Subtypes Finally, we evaluated the specificity and sensitivity of these six validated miRNAs in combination with their predicted mRNAs to discriminate between SCC and adenocarcinoma (figure 8). The best performance was observed for KRT6A, as a target of miR-375, with sensitivity and specificity values of 94.1% and 88.9%, respectively. Good diagnostic performance was also observed for CEACAM6, ACSL5 y MLPH, as targets of miR-205, with lower specificity values (71.4–76.2%) but higher sensitivity (100%). Figure 3. Experimental validation of deregulated mRNA in the training cohort. To validate genes identified as differentially expressed by tumor histology in the microarray data, relative expression levels of mRNAs were quantified by real-time PCR using the DCt method by B2M as housekeeping gene. The plots show median DCt values of validated genes in patients with adenocarcinoma versus SCC. Data derived from RT-qPCR are presented as log 2 2 2DCt values. Pvalue below 0.05 was considered significant. doi:10.1371/journal.pone.0090524.g003 Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 7 March 2014 | Volume 9 | Issue 3 | e90524 Finally, TMEMB45B, as target of miR-378, showed a sensitivity of 87.5% and a specificity of 57.7%. The other miRNA/mRNA couples revealed lower sensitivity and specificity values, although they were greater than 75% and 50% in all cases, respectively (Table S2). Discussion In this study we analyzed the mRNA and miRNA expression signatures of patients with different subtypes of NSCLC. This allowed us to construct a robust transcriptional profile of lung adenocarcinoma and SCC. Our results not only indicate the existence of a mRNA and/or miRNA expression patterns that are able to distinguish between SCC and adenocarcinoma, but also that the altered gene expression signature is partly caused by specific miRNA deregulation. First, we analyzed by two approaches the whole genome expression microarray data to minimize false positives. We examined differential gene expression levels by discriminate microarray data analysis and by the one-sample t-test. Fifty-six genes were found to be significantly deregulated by both analyses and were therefore selected for further evaluation and validation. Remarkably, several of them had been previously implicated in relevant biological processes (according to gene ontology) in lung cancer. For example, some genes of the KRT family, which were down-regulated in adenocarcinoma, are involved in several critical cell functions such as cell migration, growth and proliferation [21]. Second, miRNA profiling identified 9 miRNAs that were Figure 4. Validation of deregulated mRNA in an independent cohort. Expression of nine mRNAs was validated by real-time PCR in an independent cohort of NSCLC patients. mRNA expression levels were determined in tumor samples and paired normal lung tissue from lung cancer patients and relative expression by histological subtype was assessed. Median DDCt values were determined in the validated genes in patients with adenocarcinoma and SCC. Data derived from RT-qPCR are presented as 2 2DDCt values. Pvalue below 0.05 was considered significant. doi:10.1371/journal.pone.0090524.g004 Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 8 March 2014 | Volume 9 | Issue 3 | e90524 differentially expressed among the two histological subtypes of NSCLC studied. Six of them (miR-149, miR-205, miR-375, miR- 378, miR-422a and miR-708) were further validated in an independent cohort of NSCLC patients as biomarkers able to discriminate adenocarcinoma and SCC. To assess whether these miRNAs could be directly regulating some of the 56 deregulated genes identified, several broadly used algorithms were used. Eleven of these 56 genes (20%) were thus predicted to be putative targets of at least one of the six miRNAs found to be differentially expressed in SCC compared to adenocarcinoma. Moreover, some of the predicted target genes were regulated by more than one of these miRNAs. Finally, nine (82%) of the 11 target genes were further validated by qRT-PCR in the initial training set of 44 patients and subsequently in an independent validation cohort. Other mechanisms (i.e. epigenetic regulation) are likely involved in the regulation of the remaining differentially expressed genes. Functional studies indicate that miRNAs participate in the regulation of almost every cellular process investigated and that changes in their expression are observed in diseases such as cancer. Bioinformatic predictions indicate that mammalian miRNAs can regulate approximately 30% of all protein-coding genes [22]. Thus, post-transcriptional regulation by microRNA may be involved in the development of the different histological phenotypes of NSCLC, and these miRNA/mRNA couples identified may prove to be useful tools for diagnostic purposes or as potential novel targets for lung cancer therapy. Consistent with this, 2 of the 6 miRNAs we validated in our study (miR-205 and miR-708) had been proposed by other investigators, in combination with miR- Figure 5. Relative quantification of deregulated microRNAs in the independent validation cohort. Expression of deregulated miRNAs was evaluated in the validation cohort. MicroRNA expression levels were determined in tumor and paired normal lung tissue of lung cancer patients and relative expression by histological subtype was assessed. Median DDCt values were determined in nine miRNAs in patients with adenocarcinoma versus SCC. Data derived from RT-qPCR are presented as 2 2DDCt values. Pvalue below 0.05 was considered significant. doi:10.1371/journal.pone.0090524.g005 Transcriptional Regulation by MicroRNAs in NSCLC PLOS ONE | www.plosone.org 9 March 2014 | Volume 9 | Issue 3 | e90524