text:
Transcriptome sequencing reveals altered long intergenic non-coding RNAs in lung cancer
DOI: 10.1186/s13059-014-0429-8
© White et al.; licensee BioMed Central Ltd. 2014
Received: 15 April 2014
Accepted: 31 July 2014
Published: 13 August 2014
Long intergenic non-coding RNAs (lncRNAs) represent an emerging and under-studied class of transcripts that play a significant role in human cancers. Due to the tissue- and cancer-specific expression patterns observed for many lncRNAs it is believed that they could serve as ideal diagnostic biomarkers. However, until each tumor type is examined more closely, many of these lncRNAs will remain elusive.
Here we characterize the lncRNA landscape in lung cancer using publicly available transcriptome sequencing data from a cohort of 567 adenocarcinoma and squamous cell carcinoma tumors. Through this compendium we identify over 3,000 unannotated intergenic transcripts representing novel lncRNAs. Through comparison of both adenocarcinoma and squamous cell carcinomas with matched controls we discover 111 differentially expressed lncRNAs, which we term lung cancer-associated lncRNAs (LCALs). A pan-cancer analysis of 324 additional tumor and adjacent normal pairs enable us to identify a subset of lncRNAs that display enriched expression specific to lung cancer as well as a subset that appear to be broadly deregulated across human cancers. Integration of exome sequencing data reveals that expression levels of many LCALs have significant associations with the mutational status of key oncogenes in lung cancer. Functional validation, using both knockdown and overexpression, shows that the most differentially expressed lncRNA, LCAL1, plays a role in cellular proliferation.
Our systematic characterization of publicly available transcriptome data provides the foundation for future efforts to understand the role of LCALs, develop novel biomarkers, and improve knowledge of lung tumor biology.
Lung cancer is among the leading causes of death worldwide and accounts for greater than 150,000 deaths per year just in the United States, greater than the combination of the next three most common cancers (colon, breast and prostate) [1]. To date, lung cancer research has primarily focused on the deregulation of protein-coding genes to identify oncogenes and tumor suppressors that could serve as diagnostic and therapeutic targets, thereby missing long non-coding RNAs (lncRNAs), which have been shown to play a critical role in tumorigenesis [2],[3]. Historical focus on protein-coding genes in disease pathology is due to the relatively recent discovery of lncRNAs, the bias of previous technologies (such as microarrays) towards protein-coding genes, and the lack of sufficient datasets to identify lncRNAs in lung cancer.
As part of the ENCODE project, the GENCODE consortium manually curated 9,277 human lncRNAs [4]. However, current estimates suggest that protein-coding genes may be outnumbered by lncRNAs, many of which have yet to be discovered due to their tissue-specific expression profiles and lower expression levels than coding genes [4]. The tissue-specific nature of lncRNAs suggests they may serve as valuable clinical markers [4]-[6]. However, until we examine each tumor type more closely, many of these clinically relevant lncRNAs may remain elusive. Transcriptome sequencing, or RNA-Seq, offers an unbiased approach for annotating expressed transcripts [5], as exemplified by the discovery of approximately 1,800 unannotated lncRNAs in a cohort of 102 prostate cancer patients, of which 121 were associated with progression [6].
Although originally regarded as transcriptional noise, several well-described examples indicate that lncRNAs may be essential actors in cancer biology, typically facilitating epigenetic gene repression through chromatin-modifying complexes. Examples include the increased expression of HOTAIR in metastatic breast cancer [7], ANRIL-induced silencing of p15 in leukemia [8], and MALAT1 association with metastasis in non-small cell lung cancer [9]. In contrast to these well-described examples, however, only a fraction of lncRNAs have documented roles in tumorigenesis [10]-[12] and even fewer have been implicated in lung cancer. The most well-characterized lncRNA reported in lung cancer is MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), which is associated with high metastatic potential and poor patient prognosis in non-small cell lung cancer patients with and without metastatic tumors [9],[13]. More recent studies have found that the intronic non-coding RNA (ncRNA) lncRNA-LET plays a role in the regulation of hypoxia-mediated metastasis in squamous cell lung carcinoma [14], intronic ncRNA AK126698 confers resistance to cisplatin by targeting the Wnt pathway [15], and the lncRNA SCAL1 (smoke and cancer-associated lncRNA-1) is associated with tobacco-induced lung cancer [16]. These individual studies demonstrate the growing importance of lncRNAs in lung cancer while highlighting the need to systematically identify lncRNAs altered in lung cancer. Given the vast quantity of lncRNAs detected and still being discovered, this represents a unique research opportunity to uncover novel biomarkers and therapeutic targets, and to understand their role in tumor biology.
In our study we harnessed the unbiased view of the transcriptome offered by massively parallel next-generation sequencing platforms to explore the recently emerging class of lncRNAs in lung cancer from 197 lung squamous cell carcinoma and 370 adenocarcinoma tumors. Overall, we were able to detect over 3,000 previously unannotated lncRNAs and identify 111 lncRNAs, termed lung cancer-associated lncRNAs (LCALs), that are strongly differentially expressed between lung tumors and adjacent normal tissue. For orthogonal validation we repurposed publicly available exon array-based data coupled with experimental validation (quantitative real-time PCR (qPCR) and rapid amplification of cDNA ends (RACE)) for a subset of LCALs. To elucidate the tissue specificity of lncRNAs altered in lung cancer we conducted a meta-analysis across an additional 324 tumor and adjacent normal pairs from seven different cancers that were sequenced as part of The Cancer Genome Atlas (TCGA) project. Additionally, we incorporated exome sequencing data from TCGA to identify LCALs that were associated with commonly mutated genes. The most differentially expressed lncRNA, LCAL1, was functionally validated and determined to regulate cellular proliferation in vitro. In summary, we have systematically characterized lncRNAs that may play a critical role in lung cancer.
Identification of novel unannotated transcripts
Characterization of novel lncRNAs
To ensure that the novel candidates that we predicted did not encode proteins, we used GeneID [24] and CPAT [25] to measure (1) the protein-coding potential and (2) the ORF size in each lncRNA sequence. For comparison, genes were classified into four categories: (i) unannotated transcripts (Novel); (ii) non-coding RNAs annotated by RefSeq (Known_RNA); (iii) protein-coding genes annotated by RefSeq (mRNA); and (iv) previously annotated lncRNAs (lncRNAs) [5]. The unannotated transcripts have a lower coding potential and ORF length relative to protein-coding genes but similar coding potential to known RNA genes and recently reported lncRNAs (Figure 1B; Figure S1A,B in Additional file 2; Table S2 in Additional file 1). Additionally, the expression levels of the novel unannotated transcripts were skewed towards lower expression, which was also observed with annotated RNAs and recently discovered lncRNAs (Figure S1C in Additional file 2). In addition to expression levels, the transcript characteristics of the novel lncRNAs mimic previously reported lncRNAs. As shown in Figure 1C, the overall transcript length of the novel lncRNAs (median 1,823 nucleotides) is shorter than protein-coding genes (2,757 nucleotides; t-test, P-value <5.4 × 10-8), which is expected given the bias of lncRNAs having fewer exons than protein-coding genes (Figure 1D).
It was recently found that transposable elements significantly contributed to the origin, diversification, and regulation of lncRNAs in human and vertebrates [26],[27]. Consistent with earlier reports [26],[27], we also found that repetitive elements accounted for 30.2% of the novel lncRNAs, with the most abundant families including LINE/L1, LINE/L2, SINE/Alu, SINE/MIR, and LTR/ERVL-MaLR (Figure S1D in Additional file 2).
To determine whether the predicted novel lncRNAs are independent transcripts rather than extensions of neighboring protein-coding transcripts [28], we leveraged existing ENCODE ChIP-Seq data available for the H3K4me3 histone modification that is associated with active promoters. We focused on the epithelial cell line (A549) derived from a lung carcinoma tissue to better reflect our tumor tissue cohort. We observed enrichment for histone modifications characterizing transcriptional start sites and active transcription (Figure 1E). Protein-coding transcripts had the highest enrichment, with recently discovered lncRNAs [5] and novel lncRNAs showing nearly equivalent profiles. Taken together, characterization of the unannotated transcripts suggests that they are novel lncRNAs.
Altered lncRNA expression in lung cancer tissues relative to adjacent normal lung tissues
Using the results from all three cohorts, we composed a list of 111 intergenic lung cancer-associated lncRNAs (LCALs) that represent the most highly expressed and differentially expressed transcripts (Figure 2D; Table S6 in Additional file 1). Fifty LCALs were differentially expressed in all three cohorts, 22 in two cohorts, and 39 unique to a single cohort. Not surprisingly LUSC had the most cohort-specific lncRNAs, as it is the only squamous cell lung cancer cohort in the study. Additionally, 57 LCALs were differentially expressed in both adenocarcinoma cohorts and 21 were differentially expressed in a single adenocarcinoma cohort. The differences between the LUAD and Seo lncRNAs may represent differences in the ethnic backgrounds amongst the patient population since the Seo cohort is an exclusively Korean patient population.
The 111 LCALs include a lncRNA known to play a role in lung cancer (SCAL1[16]), cancer-associated lncRNAs not previously implicated in lung cancer (CCAT1[29], ESCCAL-1[30], LINC00261[31], linc-UBC1[32], UCA1[33], ENST00000547963 [34], and PART-1[35]), a lncRNA implicated in a lethal lung developmental disorder (FENDRR[36]), and three previously unannotated lncRNAs. Interestingly, the remaining 99 lncRNAs were previously annotated in normal human tissues but not implicated in human disease.
LncRNAs associated with lung cancer subtypes
Lung cancer is a heterogeneous disease comprised of different subtypes and molecular aberrations. Therefore, we next sought to better understand the role of lncRNAs in each subtype. We found 463 and 315 up- and down-regulated genes, respectively, in LUAD tumors relative to LUSC (Table S7 in Additional file 1; Figure S3 in Additional file 2). Of the 50 LCALs that differentiated tumor from normal tissues across all three cohorts, 27 were differentially expressed between LUAD and LUSC tumors. This subset of LCALs could potentially serve as important biomarkers for lung cancer due to their differential expression between tumor and normal lung tissue as well as between adenocarcinoma and squamous cell carcinoma tumors.
Orthogonal validation of altered lung adenocarcinoma lncRNAs using Affymetrix exon arrays
To provide additional independent validation of altered lncRNA expression, we repurposed the existing Affymetrix Human Exon 1.0 ST array with publicly available expression profiling data from an independent cohort of 20 adenocarcinoma lung cancer patient tumor and adjacent normal samples collected at the University of Pittsburgh (Gene Expression Omnibus accession GSE12236) [37]. In total, 81.25% of all lncRNAs were covered by at least one probeset overlapping an exon (including 57.9% of the 3,246 novel lncRNAs). Of the 111 LCALs, 98 (88.3%) were covered by at least one probeset. This demonstrates that although the Human Exon Array is able to measure expression levels of a large number of lncRNAs, it does not provide the same genome-wide coverage as RNA-Seq and therefore misses potentially informative lncRNAs.
Experimental validation of LCALs in cell lines and an independent tissue panel
LncRNAs are known to display features typical of transcription by RNA polymerase II, including 5′ capping, 3′ polyadenylation, and intron splicing [38]. However, despite observing H3K4me3 marks, indicative of promoter regions for the novel lncRNAs, we were still concerned that the lower expression levels of lncRNAs would poorly define the transcript boundaries. Therefore, to characterize the lncRNA transcripts and ensure that we observe the full-length transcript, we designed gene-specific primers for four lncRNA genes and conducted 5′ RACE and 3′ RACE using Invitrogen's Gene Racer Kit. In each instance we were able to recapitulate a full-length transcript corresponding to the observed RNA-Seq coverage (Figure 4; Figure S5 in Additional file 2).
Aberrantly expressed lncRNAs across human cancers
We further investigated LCALs that were altered across multiple cancers. Of the nine LCALs that were altered in at least three additional cancers, only LCAL84 has been previously studied in cancer. LCAL84 (ENST00000547963) is a member of a three-lncRNA signature associated with the survival of patients with esophageal squamous cell cancer [34]; thus, it is not unexpected that it is differentially expressed in the two squamous cell cohorts, head and neck and lung, although it is also differentially expressed in colon and stomach adenocarcinoma. Two of the experimentally validated LCALs, LCAL5 and LCAL80, were also broadly altered across three additional cancers (Figure 5C). This meta-analysis emphasizes the potential significance of previously uncharacterized lncRNAs across multiple cancers.
Associations with mutation status
Characterization of LCAL1
To determine if the lncRNAs found in this study have phenotypic consequences, we chose to examine the most differentially expressed lncRNA, LCAL1, in both adenocarcinoma and squamous cell carcinoma. LCAL1 is located on chromosome 6q14.1 and produces a three-exon transcript (Figure 4A). ENCODE data show DNaseI hypersensitivity and transcription factor binding upstream of LCAL1, suggesting regulatory activity within the LCAL1 promoter (Figure S7 in Additional file 2). Interestingly, LCAL1 lacks strong base pair conservation, using PhyloP. However, LCAL1 appears to be evolutionarily conserved amongst primates, suggesting a more recent evolution (Figure S7 in Additional file 2). Subcellular localization revealed that LCAL1 was enriched in the nucleus, which is common amongst lncRNAs associated with gene regulation [4] (Figure S8 in Additional file 2).
To confirm that changes in cell growth were due to a proliferative effect of LCAL1 expression, Alamar Blue proliferation experiments were also conducted. After 72 hours LCAL1 knockdown cells were replated and Alamar Blue reduction was assessed on days 2, 4, and 6. We see a similar significant decrease of proliferation in both siRNA constructs compared with scrambled control in both H322M and HCC95 cell lines (Figure S10 in Additional file 2). In addition, there was no change in apoptosis or necrosis in both cell lines with decreased LCAL1 expression compared with control as measured by annexin V and propidium iodide staining at 72 hours post-knockdown (data not shown). These results highlight the biological importance of LCAL1 in promoting tumorigenesis.
The utilization of lncRNAs as biomarkers, and more recently active tumorigenic factors influencing protein function, demonstrates the necessity for more extensive studies characterizing and understanding the role of lncRNAs in disease progression. In this study we used an unbiased approach to systematically categorize lncRNAs in 567 tumors from three separate publicly available RNA-Seq lung data sets. We identified 111 intergenic lung cancer-associated lncRNAs, or LCALs, most of which were not previously implicated in cancer development and progression. Further stratification of the 111 LCALs determined 27 LCALs to be subtype-specific, and therefore might serve as important biomarkers to form a molecular signature in stratifying adenocarcinoma and squamous cell carcinoma. A meta-analysis across seven additional cancers established that most (over 50%) LCALs appear to have restricted expression in lung cancer, suggesting they may be involved in disease pathogenesis and serve as putative biomarkers. Moreover, a small percentage of LCALs are highly differentially expressed in at least one other cancer, with nine being expressed in at least three additional cancers. This analysis highlights the importance of lncRNAs not only in lung cancer but also as broad oncogenic factors and lays the groundwork for future studies to determine the mechanisms by which these newly discovered non-coding RNAs act in cancer progression.
In our study we provided a comprehensive analysis to detect novel lncRNAs across lung cancer patients that led to the annotation of over 3,000 novel lncRNAs. However, to ensure that we were annotating high-confidence candidates we focused on multi-exon genes. Additionally, the publicly available data collections used for this study did not utilize stranded libraries and therefore did not allow for accurate annotation of antisense non-coding RNAs. Furthermore, the data used in this study focused on polyA+ RNA and therefore may have missed some non-coding RNAs. However, for the first time we were able to identify solid tumor-associated lncRNAs not previously implicated in lung cancer as well as uncharacterized lncRNAs altered in lung cancer. For example, linc-UBC1 (LCAL6) was discovered in bladder cancer [32]; UCA1 (LCAL52) in bladder [33], ovarian [46] and breast cancer [47]; LINC00261 (LCAL62) in gastric cancer [31]; ESCCAL-1 (LCAL80) [30] and ENST00000547963 (LCAL84) [34] in esophageal squamous cell carcinoma; CCAT1 (LCAL85) in colon cancer [29]; and PART1 (LCAL92) in prostate cancer [35] and glioblastoma multiforme [48]. Overall, these findings emphasize the importance of unbiased sequencing approaches to better understand the non-coding RNA landscape of cancer.
One of the major challenges for studying lncRNAs is to determine their potential functional role. Interestingly, mutations in well-established oncogenes have shown association with lncRNAs. For example, the lncRNA BANCR was found as a recurrently overexpressed transcript in BRAFV600E-mutant human melanoma, which is the most activating mutation in melanoma, with a potential role in regulating cell migration [43]. Additional studies have also established that lncRNAs, such as lncRNA-p21, contain functional p53-binding motifs [49], indicating these lncRNAs serve as transcriptional targets in key biological pathways. Here we discovered that some LCALs are also associated with mutational status, thereby implicating them in key oncogenic pathways. In addition to altered LCAL expression associating with TP53 mutation status, some of the LCALs also associated with mutations in KEAP1 and NFE2L2, which are key players in the oxidative stress pathway. For instance, SCAL1 (LCAL51) was found to be associated with KEAP1 mutation status in LUAD and NFE2L2 mutation status in LUSC (Figure 6B) and was recently shown to act downstream of NRF2 and mediate oxidative stress protection in airway epithelial cells [16]. The association of SCAL1 with oxidative stress, which has been previously explored through experimental validation [16], further supports that the association of LCAL expression with mutational status can potentially elucidate their function and serve as the basis for future cancer biology studies.
To determine the importance of these LCALs in lung disease pathology, we proceeded with functional studies of LCAL1, the top up-regulated lncRNA in both LUAD and LUSC. Cellular proliferation studies revealed an oncogenic phenotype, as shown by siRNA knockdown studies of LCAL1 resulting in decreased cellular growth in two cellular models of lung cancer, a non-small cell lung carcinoma cell line (H322M) and a squamous cell carcinoma cell line (HCC95). Moreover, as proof-of-principle, our LCAL1 overexpression studies highlight increased proliferation compared with control BEAS-2B empty vector cells, suggesting that altered LCAL1 is sufficient for promoting the etiology of the disease. Furthermore, our LCAL1 experiments highlight the potential functional contribution additional LCALs may have in various facets of lung tumorigenesis.
To date, lung cancer research has primarily focused on the deregulation of protein-coding and microRNA genes to identify oncogenes and tumor suppressors as potential diagnostic and therapeutic targets. However, lncRNAs represent an emerging and under-studied class of transcripts that have a significant role in human cancers. This study leverages RNA-Seq data from approximately 550 patient specimens representing an unmatched lung cancer transcriptome analysis to date to discover 111 lung cancer-associated lncRNAs (LCALs). We have experimentally validated a subset of LCALs and demonstrated that the most commonly up-regulated lncRNA across lung subtypes, LCAL1, contributes to cellular proliferation. A meta-analysis across human cancers revealed a subset of LCALs that have restricted expression and may represent putative biomarkers while a subset appear to be altered in multiple solid tumors, suggesting a common oncogenic role. Taken together, our study highlights the comprehensive scope of lncRNAs (both previously known and novel) that may contribute to lung cancer. While we already demonstrate the biological significance of LCAL1, our study provides a framework for subsequent research exploring additional LCALs in lung tumorigenesis as well as assessing their prognostic and predictive potential.
Materials and methods
Lung RNA-Seq datasets
Raw sequences from three previously sequenced lung RNA-Seq datasets were downloaded: (1) 72 adenocarcinoma tumor and adjacent normal pairs [18] (referred to as 'Seo') from EBI-SRA under accession number ERP001058; (2) 55 adenocarcinaoma tumors and adjacent normal pairs, plus an additional 243 unmatched tumors, from TCGA (referred to as 'LUAD'); and (3) 34 squamous cell carcinoma tumors and adjacent normal pairs, plus an additional 163 unmatched tumors, from TCGA [17] (referred to as 'LUSC'). Sequence reads were aligned using TopHat v1.3.0 [50].
Discovery of unannotated lncRNAs
All available samples (adjacent normal, matched tumor, unmatched tumor) from the LUAD, LUSC and Seo cohorts were used to discover novel expressed transcripts. Transcript assemblies were generated using Cufflinks v2.0.2 [19] in de novo mode and subsequently merged together with Cuffmerge to generate a consensus transcriptome across the cohort. To identify unannotated transcripts, a comprehensive set of protein-coding gene annotations was generated by downloading RefSeq, UCSC, Ensembl and GENCODE v17 gene annotations, in gene transfer format (GTF) and aggregated together (each downloaded on 20 September 2013). Additionally, the lncRNAs identified from the Human Body Map project were downloaded from UCSC and aggregated to the protein-coding GTF. Cuffcompare was used to compare the lung cancer consensus transcriptome with our comprehensive protein-coding and lncRNA gene reference. The Cuffcompare results were filtered for gene loci that were classified as unannotated (`u') and none of the transcripts overlapped an existing gene annotation. This subset was defined as `novel transcripts'. Analysis of coding potential of lncRNAs was performed on transcript sequences using GeneID [24] and CPAT and were both pre-trained for human genes [25].
Enrichment for H3K4me3 histone modifications in lung cancer cells was conducted using ENCODE ChIP-Seq data downloaded from the UCSC browser tracks. Coverage was aggregated across 500 nucleotide bins for 20 kb upstream and downstream of the transcription start site for each transcript in the following categories: (i) protein-coding, (ii) known RNAs (reported in RefSeq), (iii) lncRNAs recently annotated in human development, (iv) novel lncRNAs found in this study, and (v) random. The random transcriptional start sites were determined by selecting genomic coordinates randomly throughout the genome.
Repetitive element analysis of lncRNAs
RepeatMasker annotation for human genome (hg19 assembly) was downloaded from the UCSC database [20]. BEDTools intersect [51] was used to identify overlap between repetitive elements and transcript exons. Repetitive elements that overlapped at least 10 nucleotides with an exon were considered for further analysis. Anything that was not classified as a transposable element (such as low complexity, satellites, and simple repeats) was removed from further analysis.
Gene expression analysis
Figure S2A in Additional file 2 shows the multiple steps of our differential expression pipeline. A custom annotation file comprised of lncRNAs from multiple sources was generated by merging noncoding transcripts from GENCODE v17, Ensembl, UCSC, Human Body Map, and our novel lncRNAs. All single-exon transcripts were removed. This list was then merged with all RefSeq noncoding transcripts, including single-exon transcripts, and all transcripts less than 200 nucleotides were removed. Transcripts overlapping an exon from a RefSeq protein coding gene or Ensembl pseudogene were removed, resulting in 34,308 unique transcripts spanning 14,091 gene loci. Gene expression FPKM values were calculated with Cufflinks v2.0.2 using this custom lncRNA annotation file. Additionally, a table comprising read counts for each transcript was calculated using BEDTools version 2.17.0 [51]. We removed lowly expressed transcripts (at least 75% of samples had FPKM <0.1 or read count <25). The set of remaining transcripts was reduced to a set of non-overlapping regions (or 'genes') by comparing all overlapping transcripts and keeping the transcript with the largest average FPKM across all samples as the representative transcript for that region. After TMM normalization [52], edgeR version 3.0.8 [53] was used to identify differentially expressed transcripts between tumor and normal pairs using a matched pair design for the Seo, LUAD and LUSC datasets using cutoffs of false discovery rate (FDR) <10-5 and absolute fold change ≥2. To obtain the list of LCALs, we selected lncRNAs for which the following criteria all held across at least one cohort: (1) differentially expressed, (2) highly expressed (average tumor or normal FPKM ≥2), and (3) large fold change between tumor and normal (fold change ≥8).
The same pipeline was used for discovering differentially expressed lncRNAs between tumor subtypes. The only difference was that instead of using a matched pair design, we tested for a difference between subgroups after adjusting for gender (LUAD, n = 297; LUSC, n = 196; two samples without gender information were removed). Heatmaps were generated for each dataset using standardized values by subtracting the median and dividing by the median absolute deviation of each lncRNA. Rows (lncRNAs) were clustered using Ward's method.
Expression levels of validated LCALs
For the six LCALs that were experimentally validated (LCALs 1, 5, 7, 18, 80, and 85), coverage across the transcript was calculated by counting the read depth at each base using custom perl scripts and the Bio::DB::Sam BioPerl package. Coverage maps shown in Figure 4 and in Figure S5 in Additional file 2 were created in R using SigFuge version 1.1.2 [54]. For the tables shown in Figure 4 and in Figure S5 in Additional file 2, the cutoff for classifying samples as high or low expression was determined by maximizing the Matthews correlation coefficient [55] and two-sided P-values were calculated using Fisher's exact test.
Human exon array validation
Affymetrix Human Exon 1.0 ST Array data for 20 lung adenocarcinoma tumor and adjacent normal pairs [37] were downloaded from Gene Expression Omnibus (GSE12236). We chose to repurpose this array platform because it has the most comprehensive probe coverage of lncRNA genes. The genomic coordinates for each probeset were converted from hg18 to hg19 using the UCSC Genome Browser LiftOver tool [56]. Probesets overlapping exons of lncRNAs were determined using custom perl scripts. For annotated lncRNAs, only probesets on the correct strand were used. For the novel lncRNAs where the strand is unknown, overlapping probes on either strand were used. The raw CEL files were processed using Affymetrix Power Tools [57] with RMA normalization [58] to generate transcript-level intensity estimates. For each LCAL that was called differentially expressed in either adenocarcinoma cohort (LUAD or Seo), a paired Wilcoxon signed rank test was performed. LCALs with P < 0.05 were considered to be validated by the array.
Aberrantly expressed lncRNAs across human cancers
TCGA MapSplice aligned BAM files [59] were downloaded from TCGA for tumor and adjacent normal pairs from the following tissue types: breast invasive carcinoma (n = 104 pairs), colon adenocarcinoma (n = 16), head and neck squamous cell carcinoma (n = 37), kidney renal clear cell carcinoma (n = 69), stomach adenocarcinoma (n = 30), thyroid carcinoma (n = 58), and uterine corpus endometrial carcinoma (n = 10). A table comprising read counts for each transcript from our custom lncRNA annotation file was calculated using BEDTools version 2.17.0 [51]. Similar to the edgeR pipeline previously described, transcripts less than 200 nucleotides, with only one exon, or overlapping known pseudogenes were removed along with protein coding genes. Log fold changes were obtained from edgeR and FPKM [19] expression values were manually calculated as 109(M/(T × L)) where M is the number of reads mapping to a transcript, T is the total number of mapped reads, and L is the transcript length. LncRNAs with FDR ≤10-5, absolute fold change ≥2, altered in the same direction as lung, and either average tumor FPKM or average normal FPKM ≥1 were called significantly differentially expressed.
Association of LCALswith mutation status
The most frequently mutated genes in lung cancer were determined as having over 10% mutation rate in either the LUAD or LUSC cohort, as reported in Figure 2 from Kandoth et al.[44]. Mutation calls were downloaded from TCGA [60]. Attention was restricted to 167 LUAD and 178 LUSC samples with both RNA-Seq and mutation data. A Wilcoxon rank sum test was used to test for significance between mutational status and expression of each LCAL (using manually calculated FPKM values). For each mutated gene, P-values for the LCALs were corrected for multiple comparisons using the Benjamini and Hockberg FDR correction [61], and a significance threshold of 0.01 was used.
Cell culture and human lung cancer RNA
A549, HOP62, HOP92, NCI-H522, -H32, -H460, -H322M, and -H226 were a kind gift from Dr Van Tine at Washington University. Calu-1, SK-MES-1, SW900, and HCC95 were a kind gift from Dr Loren Michel at Washington University. HCC827 was a kind gift from Dr Leonard Maggi at Washington University. BEAS-2B cells were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were grown in RPM1-1640 (Invitrogen, Carsbad, CA, USA) with 10% fetal bovine serum and 1% penicillin/streptomycin. RNA (2 μg) from lung cancer tissue and their matched controls was obtained from the Tissue Procurement Core at Washington University.
RNA isolation and cDNA synthesis
Total RNA was isolated with the RNeasy Mini Kit (QIAGEN) with DNase 1 treatment according to the manufacturer's instructions. cDNA was synthesized from total RNA using High Capacity cDNA Reverse Transcription Kit with random hexamers (Invitrogen). Human lung cancer tissue RNA was used to make cDNA with the Superscript III RT-PCR Kit (Invitrogen).
Quantitative real-time PCR
At least two biological replicates were used for qPCR using PowerSyBr Green (Invitrogen). The comparative CT (ΔΔCT) method was used with values first normalized to the housekeeping gene RPL32, and then to BEAS-2B control. All primers were obtained from Integrated DNA Technologies (Coralville, IA, USA) and are listed in Table S9 in Additional file 1. Primer efficiency between 90 and 110% was determined for each primer candidate.
5' and 3' RACE was done using the GeneRacer Kit (Invitrogen) according to the manufacturer's instructions. RACE PCR products were obtained with Platinum Taq High Fidelity (Invitrogen) using the GeneRacer primer (supplied) and a gene-specific primer (GSP) listed in Table S10 in Additional file 1. Nested PCR was also performed for most transcripts. Products were visualized on a 2% agarose gel and purified by gel extraction (QIAGEN). This product was then cloned into pcr4-TOPO vector (Invitrogen) and grown in TOP10 Escherichia coli. Clones were sequenced with the M13 forward primer at The Protein and Nucleic Acid Chemistry Laboratory at Washington University. Full-length sequences were uploaded to GenBank under the following accession numbers: KF773845 (LCAL1), KF773846 (LCAL5), KF773847 (LCAL7), and KF773848 (LCAL80).
siRNA knockdown experiments
Stealth siRNA oligonucleotides were synthesized by Invitrogen. The following siRNA sequences were used for knockdown of LCAL1: siRNA 1 GGACAGGCTGCAGTCATCATATGGA and siRNA 2 GGCATGTGTTCAGACATATCCTAAA. Cells were transfected with 50 pmol of siRNA and a scrambled-matched %GC oligo as control with RNAimax Lipofecatmine (Invitrogen) following the manufacturer's instructions. Knockdown efficiency was determined by qPCR at time of plating for assay. After 72 hours, cells were then plated at 200,000 cells/well for proliferation assays. Cells were counted using the Beckman Z1 Coulter Counter at days 2, 4, and 6. At least three biological replicates were performed for each siRNA construct over two experiments.
Alamar Blue proliferation assays
Seventy-two hours after transfection, cells were seeded at 3,000 cells/well in a 96-well dish to assess viability via Alamar Blue according to the manufacturer's instructions (Sigma). Subsequent cells were then used for RNA isolation to detect relative expression of LCAL1. Fluorescence intensity was then measured with Gen5 software on Synergy Hybrid (BioTek) at days 2, 4, and 6 post-knockdown after one hour incubation with Alamar Blue. At least four biological replicates were done for each siRNA construct over two experiments.
Retroviral infection and generation of BEAS-2B cell lines stably expressing LCAL1 variants
The full length LCAL1 transcript was PCR amplified from H322M cells and cloned into the pCFG5-IEGZ vector (a kind gift from Dr Ron Bose). Full-length inserts were confirmed with Sanger sequencing at The Protein and Nucleic Acid Chemistry Laboratory at Washington University. Retroviral infection of BEAS-2B cells were performed according to Kavuri et al. [62]. Briefly, the amphotrophic producer cell lines were transfected with 10 μg of LCAL1 and empty control retroviral vectors by calcium phosphate precipitation and incubated for 24 hours. Viral supernatants were harvested after an additional 24 hour incubation. Virus was added to BEAS-2B cells seeded in six-well dishes in the presence of 8 μg/ml Polybrene. BEAS-2B cells were centrifuged at 2,500 RPM for 1.5 hours at 22°C and supernatant exchanged for fresh media. After 10 to 14 days of 125 μg/ml zeocin selection, cells were plated at 200,000 cells/well for proliferation assays. Cells were counted using the Beckman Z1 Coulter Counter at days 2, 4, and 6. At least three biological replicates were performed for each stable cell line over two experiments. Cells were also collected for validation of LCAL1 expression by qPCR.
Nuclear localization
H322M lysates were fractionated into nuclear and cytosolic fractions according to the PARIS kit protocol (Invitrogen) and gene expression was assessed by qPCR. Results were normalized to the housekeeping gene RPL32, and then to total RNA. U6 was used as a positive control for nuclear gene expression and GAPDH and MT-RNR1 were used as positive cytoplasmic gene expression. Three biological replicates were conducted over two independent experiments.
Additional files
- ChIP-Seq:
chromatin immunoprecipitation sequencing
false discovery rate
fragments per kilobase of transcript per million mapped reads
gene transfer format
lung cancer-associated lncRNA
- lncRNA:
long non-coding RNA
lung adenocarcinoma
lung squamous cell carcinoma
open reading frame
- qPCR:
quantitative real-time PCR
rapid amplification of cDNA ends
- siRNA:
short interfering RNA
The Cancer Genome Atlas
This work was partially funded by a LUNGevity Career Development Award and American Lung Association Biomedical Research Grant (CAM). This work was supported by the National Human Genome Research Institute (NHGRI) U54 HG003079 (PI: Richard K Wilson).
Authors' Affiliations
- Jemal A, Siegel R, Xu J, Ward E: Cancer statistics, 2010. CA Cancer J Clin. 2010, 60: 277-300. 10.3322/caac.20073.PubMedView ArticleGoogle Scholar
- Prensner JR, Chinnaiyan AM: The emergence of lncRNAs in cancer biology. Cancer Discov. 2011, 1: 391-407. 10.1158/2159-8290.CD-11-0209.PubMedPubMed CentralView ArticleGoogle Scholar
- Gutschner T, Diederichs S: The hallmarks of cancer: a long non-coding RNA point of view. RNA Biol. 2012, 9: 703-719. 10.4161/rna.20481.PubMedPubMed CentralView ArticleGoogle Scholar
- Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, Guernec G, Martin D, Merkel A, Knowles DG, Lagarde J, Veeravalli L, Ruan X, Ruan Y, Lassmann T, Carninci P, Brown JB, Lipovich L, Gonzalez JM, Thomas M, Davis CA, Shiekhattar R, Gingeras TR, Hubbard TJ, Notredame C, Harrow J, Guigó R: The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression. Genome Res. 2012, 22: 1775-1789. 10.1101/gr.132159.111.PubMedPubMed CentralView ArticleGoogle Scholar
- Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, Rinn JL: Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011, 25: 1915-1927. 10.1101/gad.17446611.PubMedPubMed CentralView ArticleGoogle Scholar
- Prensner JR, Iyer MK, Balbin OA, Dhanasekaran SM, Cao Q, Brenner JC, Laxman B, Asangani IA, Grasso CS, Kominsky HD, Cao X, Jing X, Wang X, Siddiqui J, Wei JT, Robinson D, Iyer HK, Palanisamy N, Maher CA, Chinnaiyan AM: Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression. Nat Biotechnol. 2011, 29: 742-749. 10.1038/nbt.1914.PubMedPubMed CentralView ArticleGoogle Scholar
- Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai M-C, Hung T, Argani P, Rinn JL, Wang Y, Brzoska P, Kong B, Li R, West RB, van de Vijver MJ, Sukumar S, Chang HY: Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010, 464: 1071-1076. 10.1038/nature08975.PubMedPubMed CentralView ArticleGoogle Scholar
- Yu W, Gius D, Onyango P, Muldoon-Jacobs K, Karp J, Feinberg AP, Cui H: Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature. 2008, 451: 202-206. 10.1038/nature06468.PubMedPubMed CentralView ArticleGoogle Scholar
- Ji P, Diederichs S, Wang W, Böing S, Metzger R, Schneider PM, Tidow N, Brandt B, Buerger H, Bulk E, Thomas M, Berdel WE, Serve H, Müller-Tidow C: MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer. Oncogene. 2003, 22: 8031-8041. 10.1038/sj.onc.1206928.PubMedView ArticleGoogle Scholar
- Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY: Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 2007, 129: 1311-1323. 10.1016/j.cell.2007.05.022.PubMedPubMed CentralView ArticleGoogle Scholar
- Matouk IJ, DeGroot N, Mezan S, Ayesh S, Abu-lail R, Hochberg A, Galun E: The H19 non-coding RNA is essential for human tumor growth. PLoS ONE. 2007, 2: e845-10.1371/journal.pone.0000845.PubMedPubMed CentralView ArticleGoogle Scholar
- Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, Xiong Y: Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene. Oncogene. 2011, 30: 1956-1962. 10.1038/onc.2010.568.PubMedPubMed CentralView ArticleGoogle Scholar
- Gutschner T, Hämmerle M, Eissmann M, Hsu J, Kim Y, Hung G, Revenko A, Arun G, Stentrup M, Gross M, Zörnig M, MacLeod AR, Spector DL, Diederichs S: The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res. 2013, 73: 1180-1189. 10.1158/0008-5472.CAN-12-2850.PubMedPubMed CentralView ArticleGoogle Scholar
- Yang F, Huo X, Yuan S, Zhang L, Zhou W, Wang F, Sun S: Repression of the long noncoding RNA-LET by histone deacetylase 3 contributes to hypoxia-mediated metastasis. Mol Cell. 2013, 49: 1083-1096. 10.1016/j.molcel.2013.01.010.PubMedView ArticleGoogle Scholar
- Yang Y, Li H, Hou S, Hu B, Liu J, Wang J: The noncoding RNA expression profile and the effect of lncRNA AK126698 on cisplatin resistance in non-small-cell lung cancer cell. PloS One. 2013, 8: e65309-10.1371/journal.pone.0065309.PubMedPubMed CentralView ArticleGoogle Scholar
- Thai P, Statt S, Chen CH, Liang E, Campbell C, Wu R: Characterization of a novel long noncoding RNA, SCAL1, induced by cigarette smoke and elevated in lung cancer cell lines. Am J Respir Cell Mol Biol. 2013, 49: 204-211. 10.1165/rcmb.2013-0159RC.PubMedPubMed CentralView ArticleGoogle Scholar
- Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012, 489: 519-525. 10.1038/nature11404.
- Seo JS, Ju YS, Lee WC, Shin JY, Lee JK, Bleazard T, Lee J, Jung YJ, Kim JO, Shin JY, Yu SB, Kim J, Lee ER, Kang CH, Park IK, Rhee H, Lee SH, Kim JI, Kang JH, Kim YT: The transcriptional landscape and mutational profile of lung adenocarcinoma. Genome Res. 2012, 22: 2109-2119. 10.1101/gr.145144.112.PubMedPubMed CentralView ArticleGoogle Scholar
- Trapnell C, Williams B, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010, 28: 511-515. 10.1038/nbt.1621.PubMedPubMed CentralView ArticleGoogle Scholar
- Meyer LR, Zweig AS, Hinrichs AS, Karolchik D, Kuhn RM, Wong M, Sloan CA, Rosenbloom KR, Roe G, Rhead B, Raney BJ, Pohl A, Malladi VS, Li CH, Lee BT, Learned K, Kirkup V, Hsu F, Heitner S, Harte RA, Haeussler M, Guruvadoo L, Goldman M, Giardine BM, Fujita PA, Dreszer TR, Diekhans M, Cline MS, Clawson H, Barber GP, et al: The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res. 2013, 41: D64-D69. 10.1093/nar/gks1048.PubMedPubMed CentralView ArticleGoogle Scholar
- Flicek P, Ahmed I, Amode MR, Barrell D, Beal K, Brent S, Carvalho-Silva D, Clapham P, Coates G, Fairley S, Fitzgerald S, Gil L, García-Girón C, Gordon L, Hourlier T, Hunt S, Juettemann T, Kähäri AK, Keenan S, Komorowska M, Kulesha E, Longden I, Maurel T, McLaren WM, Muffato M, Nag R, Overduin B, Pignatelli M, Pritchard B, Pritchard E, et al: Ensembl 2013. Nucleic Acids Res. 2013, 41: D48-D55. 10.1093/nar/gks1236.PubMedPubMed CentralView ArticleGoogle Scholar
- Harrow J, Frankish A, Gonzalez JM, Tapanari E, Diekhans M, Kokocinski F, Aken BL, Barrell D, Zadissa A, Searle S, Barnes I, Bignell A, Boychenko V, Hunt T, Kay M, Mukherjee G, Rajan J, Despacio-Reyes G, Saunders G, Steward C, Harte R, Lin M, Howald C, Tanzer A, Derrien T, Chrast J, Walters N, Balasubramanian S, Pei B, Tress M, et al: GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012, 22: 1760-1774. 10.1101/gr.135350.111.PubMedPubMed CentralView ArticleGoogle Scholar
- Pruitt KD, Brown GR, Hiatt SM, Thibaud-Nissen F, Astashyn A, Ermolaeva O, Farrell CM, Hart J, Landrum MJ, McGarvey KM, Murphy MR, O'Leary NA, Pujar S, Rajput B, Rangwala SH, Riddick LD, Shkeda A, Sun H, Tamez P, Tully RE, Wallin C, Webb D, Weber J, Wu W, DiCuccio M, Kitts P, Maglott DR, Murphy TD, Ostell JM: RefSeq: an update on mammalian reference sequences. Nucleic Acids Res. 2014, 42: D756-D763. 10.1093/nar/gkt1114.PubMedPubMed CentralView ArticleGoogle Scholar
- Blanco E, Abril JF: Computational gene annotation in new genome assemblies using GeneID. Methods Mol Biol. 2009, 537: 243-261. 10.1007/978-1-59745-251-9_12.PubMedView ArticleGoogle Scholar
- Wang L, Park HJ, Dasari S, Wang S, Kocher JP, Li W: CPAT: Coding-Potential Assessment Tool using an alignment-free logistic regression model. Nucleic Acids Res. 2013, 41: e74-10.1093/nar/gkt006.PubMedPubMed CentralView ArticleGoogle Scholar
- Kelley D, Rinn J: Transposable elements reveal a stem cell-specific class of long noncoding RNAs. Genome Biol. 2012, 13: R107-10.1186/gb-2012-13-11-r107.PubMedPubMed CentralView ArticleGoogle Scholar
- Kapusta A, Kronenberg Z, Lynch VJ, Zhuo X, Ramsay L, Bourque G, Yandell M, Feschotte C: Transposable elements are major contributors to the origin, diversification, and regulation of vertebrate long noncoding RNAs. PLoS Genet. 2013, 9: e1003470-10.1371/journal.pgen.1003470.PubMedPubMed CentralView ArticleGoogle Scholar
- Van Bakel H, Nislow C, Blencowe BJ, Hughes TR: Most 'dark matter' transcripts are associated with known genes. PLoS Biol. 2010, 8: e1000371-10.1371/journal.pbio.1000371.PubMedPubMed CentralView ArticleGoogle Scholar
- Nissan A, Stojadinovic A, Mitrani-Rosenbaum S, Halle D, Grinbaum R, Roistacher M, Bochem A, Dayanc BE, Ritter G, Gomceli I, Bostanci EB, Akoglu M, Chen Y-T, Old LJ, Gure AO: Colon cancer associated transcript-1: a novel RNA expressed in malignant and pre-malignant human tissues. Int J Cancer J Int Cancer. 2012, 130: 1598-1606. 10.1002/ijc.26170.View ArticleGoogle Scholar
- Cao W, Wu W, Shi F, Chen X, Wu L, Yang K, Tian F, Zhu M, Chen G, Wang W, Biddle FG, Gu J: Integrated analysis of long noncoding RNA and coding RNA expression in esophageal squamous cell carcinoma. Int J Genomics. 2013, 2013: 1-10. 10.1155/2013/480534.View ArticleGoogle Scholar
- Cao W-J, Wu H-L, He B-S, Zhang Y-S, Zhang Z-Y: Analysis of long non-coding RNA expression profiles in gastric cancer. World J Gastroenterol. 2013, 19: 3658-3664. 10.3748/wjg.v19.i23.3658.PubMedPubMed CentralView ArticleGoogle Scholar
- He W, Cai Q, Sun F, Zhong G, Wang P, Liu H, Luo J, Yu H, Huang J, Lin T: linc-UBC1 physically associates with polycomb repressive complex 2 (PRC2) and acts as a negative prognostic factor for lymph node metastasis and survival in bladder cancer. Biochim Biophys Acta. 2013, 1832: 1528-1537. 10.1016/j.bbadis.2013.05.010.PubMedView ArticleGoogle Scholar
- Wang X-S, Zhang Z, Wang H-C, Cai J-L, Xu Q-W, Li M-Q, Chen Y-C, Qian X-P, Lu T-J, Yu L-Z, Zhang Y, Xin D-Q, Na Y-Q, Chen W-F: Rapid identification of UCA1 as a very sensitive and specific unique marker for human bladder carcinoma. Clin Cancer Res. 2006, 12: 4851-4858. 10.1158/1078-0432.CCR-06-0134.PubMedView ArticleGoogle Scholar
- Li J, Chen Z, Tian L, Zhou C, He MY, Gao Y, Wang S, Zhou F, Shi S, Feng X, Sun N, Liu Z, Skogerboe G, Dong J, Yao R, Zhao Y, Sun J, Zhang B, Yu Y, Shi X, Luo M, Shao K, Li N, Qiu B, Tan F, Chen R, He J: LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma. Gut. 2014, -doi:10.1136/gutjnl-2013-305806Google Scholar
- Lin B, White JT, Ferguson C, Bumgarner R, Friedman C, Trask B, Ellis W, Lange P, Hood L, Nelson PS: PART-1: a novel human prostate-specific, androgen-regulated gene that maps to chromosome 5q12. Cancer Res. 2000, 60: 858-863.PubMedGoogle Scholar
- Szafranski P, Dharmadhikari AV, Brosens E, Gurha P, Kolodziejska KE, Zhishuo O, Dittwald P, Majewski T, Mohan KN, Chen B, Person RE, Tibboel D, de Klein A, Pinner J, Chopra M, Malcolm G, Peters G, Arbuckle S, Guiang SF, Hustead VA, Jessurun J, Hirsch R, Witte DP, Maystadt I, Sebire N, Fisher R, Langston C, Sen P, Stankiewicz P: Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder. Genome Res. 2013, 23: 23-33. 10.1101/gr.141887.112.PubMedPubMed CentralView ArticleGoogle Scholar
- Xi L, Feber A, Gupta V, Wu M, Bergemann AD, Landreneau RJ, Litle VR, Pennathur A, Luketich JD, Godfrey TE: Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer. Nucleic Acids Res. 2008, 36: 6535-6547. 10.1093/nar/gkn697.PubMedPubMed CentralView ArticleGoogle Scholar
- Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, Cabili MN, Jaenisch R, Mikkelsen TS, Jacks T, Hacohen N, Bernstein BE, Kellis M, Regev A, Rinn JL, Lander ES: Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature. 2009, 458: 223-227. 10.1038/nature07672.PubMedPubMed CentralView ArticleGoogle Scholar
- Comprehensive molecular portraits of human breast tumours. Nature. 2012, 490: 61-70. 10.1038/nature11412.
- Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012, 487: 330-337. 10.1038/nature11252.
- Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013, 499: 43-49. 10.1038/nature12222.
- Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, Robertson AG, Pashtan I, Shen R, Benz CC, Yau C, Laird PW, Ding L, Zhang W, Mills GB, Kucherlapati R, Mardis ER, Levine DA: Integrated genomic characterization of endometrial carcinoma. Nature. 2013, 497: 67-73. 10.1038/nature12113.PubMedView ArticleGoogle Scholar
- Flockhart RJ, Webster DE, Qu K, Mascarenhas N, Kovalski J, Kretz M, Khavari PA: BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration. Genome Res. 2012, 22: 1006-1014. 10.1101/gr.140061.112.PubMedPubMed CentralView ArticleGoogle Scholar
- Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF, Wyczalkowski MA, Leiserson MDM, Miller CA, Welch JS, Walter MJ, Wendl MC, Ley TJ, Wilson RK, Raphael BJ, Ding L: Mutational landscape and significance across 12 major cancer types. Nature. 2013, 502: 333-339. 10.1038/nature12634.PubMedPubMed CentralView ArticleGoogle Scholar
- Ohta T, Iijima K, Miyamoto M, Nakahara I, Tanaka H, Ohtsuji M, Suzuki T, Kobayashi A, Yokota J, Sakiyama T, Shibata T, Yamamoto M, Hirohashi S: Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth. Cancer Res. 2008, 68: 1303-1309. 10.1158/0008-5472.CAN-07-5003.PubMedView ArticleGoogle Scholar
- Liu S-P, Yang J-X, Cao D-Y, Shen K: Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials. Cancer Biol Med. 2013, 10: 138-141.PubMedPubMed CentralGoogle Scholar
- Huang J, Zhou N, Watabe K, Lu Z, Wu F, Xu M, Mo Y-Y: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1). Cell Death Dis. 2014, 5: e1008-10.1038/cddis.2013.541.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang X-Q, Sun S, Lam K-F, Kiang KM-Y, Pu JK-S, Ho AS-W, Lui W-M, Fung C-F, Wong T-S, Leung GK-K: A long non-coding RNA signature in glioblastoma multiforme predicts survival. Neurobiol Dis. 2013, 58: 123-131. 10.1016/j.nbd.2013.05.011.PubMedView ArticleGoogle Scholar
- Huarte M, Guttman M, Feldser D, Garber M, Koziol MJ, Kenzelmann-Broz D, Khalil AM, Zuk O, Amit I, Rabani M, Attardi LD, Regev A, Lander ES, Jacks T, Rinn JL: A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell. 2010, 142: 409-419. 10.1016/j.cell.2010.06.040.PubMedPubMed CentralView ArticleGoogle Scholar
- Trapnell C, Pachter L, Salzberg SL: TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009, 25: 1105-1111. 10.1093/bioinformatics/btp120.PubMedPubMed CentralView ArticleGoogle Scholar
- Quinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010, 26: 841-842. 10.1093/bioinformatics/btq033.PubMedPubMed CentralView ArticleGoogle Scholar
- Robinson MD, Oshlack A: A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010, 11: R25-10.1186/gb-2010-11-3-r25.PubMedPubMed CentralView ArticleGoogle Scholar
- Robinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010, 26: 139-140. 10.1093/bioinformatics/btp616.PubMedPubMed CentralView ArticleGoogle Scholar
- SigFuge. [http://www.bioconductor.org/packages/devel/bioc/html/SigFuge.html]
- Matthews B: Comparison of the predicted and observed secondary structure of T4 phage lysozyme. Biochim Biophys Acta. 1975, 405: 442-451. 10.1016/0005-2795(75)90109-9.PubMedView ArticleGoogle Scholar
- UCSC Genome Browser LiftOver. [http://genome.ucsc.edu/cgi-bin/hgLiftOver]
- Affymetrix Power Tools. [http://www.affymetrix.com/partners_programs/programs/developer/tools/powertools.affx]
- Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003, 4: 249-264. 10.1093/biostatistics/4.2.249.PubMedView ArticleGoogle Scholar
- Wang K, Singh D, Zeng Z, Coleman SJ, Huang Y, Savich GL, He X, Mieczkowski P, Grimm SA, Perou CM, MacLeod JN, Chiang DY, Prins JF, Liu J: MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res. 2010, 38: e178-10.1093/nar/gkq622.PubMedPubMed CentralView ArticleGoogle Scholar
- TCGA Pan-cancer MAF files. [http://www.synapse.org/#!Synapse:syn1729383]
- Benjamini Y, Hochberg Y: Controlling the false discovery rate- a practical and powerful approach to multiple testing. J R Stat Soc Ser B. 1995, 57: 289-300.Google Scholar
- Kavuri SM, Geserick P, Berg D, Dimitrova DP, Feoktistova M, Siegmund D, Gollnick H, Neumann M, Wajant H, Leverkus M: Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex. J Biol Chem. 2011, 286: 16631-16646. 10.1074/jbc.M110.148585.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


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Eurotel Victoria
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TripAdvisor Reviews Eurotel Victoria Villars-sur-Ollon
Travel Blogs from Villars-sur-Ollon
Wales and England 16-30 Oct
Wales and England - AMAZING. We arrived on Friday 16th October. We launched straight into sightseeing mode and went directly to the Roman Baths in Bath. What a groovy town Bath is. The next day was the Dr Who Experience in Cardiff. This probably rated as James' most favourite tourist attraction. That afternoon Paul and James went to the Semi Final of New Zealand v France. Our country v The Country we live in. The rest is history. ...
Food Markets!
... local food (and some from Northern Italy) is offered regardless of the weather. It's raucous and loud, with plenty of meats, cheeses, fruits and vegetables offered according to the season and the prevailing harvest.Unfortunately food seldom lasts a week (for some reason French carrots only last a few days) and come Monday I find myself obliged to return to see the lovely ladies behind the Fromager counter in Les Houches and linger some more before their ...
Quarks and Fondue
... So the material de'Nimes became known as
Avignon has been on my must see list for quite a while. It's a beautifully preserved
Medieval town and is also famous for the Pont d'Avignon, or what's left of it.
Being on the ball as I am, I decided to fully read the Rough guide when I was
halfway there. That's when I read that July is when the town hosts a drama
festival and 200,000 people attend, making normal sightseeing pretty ...
Travel pharmacy / Farmaciola
... Hayfever; Cetirizine Hydrochloride
a. Eye Allergy reaction: Dexametasona
b. Ear infection pain: Gentadexa
P. Mosquito Repellent:
a. clothing spray
Q. Sore Throat: Angina and Hibitane
R. Sea Sickness: Biodramina con cafe�na , Homeopatic pills (3 times a day, 3 ...
Here, now
... track of time, that transport me into a paralel dimension. The one of the traveler, where past lose all importance and future is just what the present will bring a day later. Found myself going from coach to coach with another couchsurfing in a place called Villars-sur-ollon, near Montreux in the French Switzerland, and I arrived that day in the most beautiful place I've seen in this trip and probably in my ...

subset: commoncrawl


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In the unfortunate case that disaster strikes, time is of the essence.
A huge 90% of SMS are read within 3 minutes of delivery which makes SMS the number one communication
tool to distribute an urgent message 'en masse'.
Other ways SMS have been used within the emergency sector include:
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Houston Texans
With Ed Reed sidelined, rookie safety D.J. Swearinger has gotten extra reps during organized team activities (OTAs) and has already made an impression on teammates, write the Houston Chronicle's Tania Ganguli and Corey Roepken. "Every day he comes out here and does something amazing," safety Danieal Manning said.
Left tackle Andrew Gardner will get to show what he can do with the first team during next week's minicamp, write Ganguli and Roepken. Starter Duane Brown had a bone spur removed from his foot Wednesday and is expected to be out for a month.
A photo gallery of Texans OTAs from the Chronicle.
Coach Gary Kubiak singled out rookies Swearinger and DeAndre Hopkins for praise after wrapping up OTAs Thursday.
Indianapolis Colts
Former Giants running back Ahmad Bradshaw visited the Colts on Thursday and the two sides have been discussing a contract, reports the Indianapolis Star's Mike Chappell. The Colts are committed to running the football more effectively a season after averaging just 3.8 yards per attempt, writes Chappell.
Reggie Wayne, who spends much of the offseason in South Florida working out, is back in Indianapolis for OTAs and next week's mandatory minicamp, AP reports. "Reggie looks great," quarterback Andrew Luck said. "Reggie's going to look great in whatever offense you put him in. If it's the spread or whatever that crazy A-11 thing was in high school a couple of years ago, whether it's backyard football, any sport. He's the type of guy that stands out. He's just an athlete. He knows how to play. He'll be great. He always is."
Jacksonville Jaguars
The Jaguars worked out free agent running back Beanie Wells, a 2009 first-round pick of the Cardinals who has struggled to stay healthy since entering the league, reports Ryan O'Halloran of the Florida Times-Union.
Uche Nwaneri played last season with torn knee cartilage and a torn meniscus, but the guard said he never considered taking himself out of the lineup to get it fixed, writes the Times-Union's Vito Stellino. "We're in the trenches. You can't show any signs of weakness. I'd do it over again in a heartbeat. A lot of it has to do with pride. At the same time, we're all in this together. The guy next to me is relying on me. I don't want to let that person down," Nwaneri said. In the offseason, Nwaneri first had arthroscopic surgery on the knee, then later had stem cells taken out of his hip and injected into his knee to regrow cartilage.
Tennessee Titans
The team waived center Eugene Amano after nine seasons with the club, AP reports.
The Titans' decision to ditch last season's playbook and start over was a gamble, but a necessary one, writes The Tennessean's David Climer.
The Titans signed Moise Fokou as a free agent this offseason, and the middle linebacker is making a case to be the starter during OTAs, writes The Tennessean's John Glennon. "He's here, in his mind, to start. That's how it should be," coach Mike Munchak said.


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This was an interesting week in the NBA's MVP race.
LeBron James has fallen victim to his own expectations, and has his eyes on a bigger prize. Kevin Durant wants this MVP more, and it has become evident. Tomorrow night OKC visits Miami and you should expect to see Durant come out victorious again as the Thunder are playing the best basketball they have ever played as a team, winning six straight against teams like the Clippers, Heat, Lakers and Bulls (before Monday's loss against Memphis).
Miami knows the regular season is pretty much meaningless to a team with championship aspirations. They have tunnel vision on that Larry O'Brien trophy. Dwyane Wade and LeBron are pacing themselves right now, catching breath before the playoffs, while Russell Westbrook and Durant haved turned it up to the max. So Durant and Westbrook have taken the advantage over LeBron and Wade respectively this week.
Durant hasn't won an MVP yet, so he wants it now more than ever. On top of that, he deserves it more than ever, so look for him to really make strides in this race while LeBron slowly, but surely falls behind.
The competition heats up in our fifth edition of our weekly "Race to the MVP" Watch.
*** *** ***
10. DERRICK ROSE – Last week: N/A
Last Week: No. 10, maintaining
Despite Rose being out for the past few weeks, there's no way you can leave him out of the top 10 MVP candidates. This season he's done enough to give the Bulls the best record in the league. That's MVP-worthy in it's own right. He expects to return before the playoffs begin. Depending on where the Bulls finish, their record ultimately decide where he finishes in this race. If they finish first, there really isn't any way you can see Rose finishing out of the top five.
9. TONY PARKER – Last week: 17.3 ppg/7.3 apg/5.0 rpg
LW: No. 9, maintaining
Tony Parker has continued to keep up his consistent level of play. As a result, the Spurs have only lost three games in the last two months (February and March). Parker has been able to contribute consistently without taking a large amount of shots. He took 20 shots or more in only two games last month, and he's been able to maintain a positive output despite nursing his injuries. The Spurs are winners of six straight so TP has a tight grip on his spot in this MVP race.
8. CHRIS PAUL – Last Week: 21.5 ppg/7.3 apg/5.0 rpg
LW: No. 8, maintaining
The Clippers seem to have finally found their niche. They've won their last six games and are right there with the Thunder and the Spurs as the hottest teams in the West. Chris Paul never accepted that role as the number one scorer for the Clippers, and that's finally started to translate into wins. In three of his four games last week, Paul had a double-double. On top of that, his turnover numbers (1.2 a game) were extremely low last week. Paul has stayed true to his point guard duties and has continued to run the team the way he planned: low shot attempts for himself, with high assist and low turnover numbers.
7. KOBE BRYANT – Last week: 21.3 ppg/5.6 rpg/4.0 apg
LW: No. 7, maintaining
Last week, Kobe Bryant continued shooting a horrendous percentage. In a win at Golden State on Tuesday he was 9-for-24 with 30 points. In a loss against OKC at home on Thursday he shot 7-for-25 with 23 points. Then against New Orleans at home he was 3-for-21 with 11 points. Eleven points on 21 attempts is not the typical efficiency of an MVP candidate. Despite his poor shooting however, the Lakers have still been good enough, losing only six games in the month of March. Kobe is still amongst the league's most valuable to his team because of what he offers in terms of leadership. But on paper, his numbers just aren't MVP worthy.
6. DWIGHT HOWARD – Last week: 18.0 ppg/10.0 rpg/1.0 bpg
LW: No. 4, Falling
Although Dwight Howard didn't play on Sunday against Denver for the Magic due to back spasms, he still had a decent week. First he put up 23 and 10 in a win against Toronto. He played terribly in that 22-point loss at New York on Wednesday, but bounced back by putting up 19 and 15 in a loss against Dallas. Two double doubles with a 1-2 record over three games… when his back spasms ease up, hopefully he can help the Magic can get back on track.
5. KEVIN LOVE – Last week: 29.0 ppg/12.5 rpg/3.3 apg
LW: No. 6, Rising
Putting up monster numbers seems to come easy to Kevin Love. It's expected of him. He knows that unless he continues to operate at a ridiculously productive level, his team won't be able to get to where he wants them to be. Kevin Love is a winner, and you can tell how badly he wants to pay for a winning team. He's been doing all he can individually to win the way he wants to. It just hasn't worked out for him yet. The Timberwolves are a good 4.5 games behind the eighth seed right now, and unless they win out, they won't have a legitimate shot at making the playoffs. However, you still can't ignore Kevin Love's individual contributions. If your definition of "most valuable" had nothing to do with team wins, he'd probably be your MVP.
4. DWYANE WADE – Last week: 21.2 ppg/5.0 apg/5.5 rpg
LW: No. 3, Falling
D-Wade has been solid for Miami, however this past week he didn't play as well as he had been. It looked as if Miami, and Wade especially, were kicking it into high gear tuning up for the playoffs. However this week it looked the exact opposite. Wade seems a bit burnt out; he isn't running the floor as much as he was, isn't defending with that pesky tenacity he was defending with. His energy seems a bit low. On paper you can't really tell.
He's continued to contribute despite taking a bit of a breather. He played great at Toronto on Friday with 30 points on 12-for-19 shooting, but aside from that it was pretty much cruise control for Wade last week.
3. RUSSELL WESTBROOK – Last week: 31.6 ppg/6.3 apg/2.3 rpg
LW: No. 5, rising
Nobody can tell Russell Westbrook anything right now. America became so infatuated with Kevin Durant's skill and humility that many of us automatically feel he has to take the most shots every night. Russell Westbrook comes off as cocky and a bit full of himself on the court, but he usually backs it up pretty well. Who can tell him he's taking too many shots when he's making most of them and the team is playing at an all-time high? Yes he's the point guard, and yes his job is to make sure the best player gets the most shots, but who's to say he isn't the best player on some nights?
When he's playing at the level he's capable of playing at and shooting a high percentage from the field, he isn't taking away from the team's success. He isn't taking food off of KD's plate. He's a matchup nightmare for some point guards; how can you criticize him for taking advantage of that? He only hurts the team when he shoots poorly and often, which has now become less often. The Thunder are playing the league's best basketball right now and he has a lot to do with it.
2. LEBRON JAMES – Last week: 18.0 ppg/7.6 apg/5.6 apg
LW: No. 1, Falling
No player in the NBA has it as difficult as LeBron James does. Quite frankly, whatever he does in the regular season will ultimately go unnoticed if Miami doesn't get the job done this year. MVP or no MVP. He knows that and he's been playing like it, just not as hard recently as we're accustomed to seeing. His effort hasn't been the same on defense and on offense he's been very passive, especially against Boston on Sunday.
Miami has been looking like a team that's tired of playing meaningless games. It almost seems as if they like their chances better as the No. 2 seed. LeBron really needs a convincing performance against OKC tomorrow night if he wants to become the leader in this race again. If Durant comes out victorious again, he really becomes the runaway favorite to win it.
1. KEVIN DURANT – Last week: 24.0 ppg/9.0 rpg/3.3 apg
LW: No. 2, Rising
KD has taken over this MVP race. Here's why: The Oklahoma City Thunder are the hottest team in the league right now. With Rose being out, Chicago has been struggling, and Miami is appearing to have it in cruise control. They look comfortable with the two seed while the Thunder appear to be playing for the league's best record. They've turned it up while Miami and Chicago have turned it down.
The Thunder have emerged as the best team in the league after recently stomping Chicago (without Rose) and Miami, and Durant has been playing consistently efficient while LeBron has been sporadic. Right now, there's no question Durant has taken over as the leader in this MVP race.
Who are your top 10 MVP candidates right now?
Follow Jaimie on Twitter at @SubZEROxPaste.
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Become a fan of Dime Magazine on Facebook HERE.


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Blt pizza
Udforsk beslægtede emner
BLT Pizza - double sauce portion of recipe to go with my homemade dough!
Ranch BLT Pizza | Ridiculously fast and easy and SUPER delicious!
BLT Pizza with White Sauce | MyRecipes.com ~ Would use turkey bacon...
BLT Pizza! Delicious, but use only 1-2 TBSP Mayo, like it suggests, I added another TBSP because I love mayo and it was way too much.
Confessions of a Skinny Girl in Training: Day 1: BLT Pizza
BLT Pizza...similar to an old favorite
BLT Pizza - double sauce portion of recipe to go with my homemade dough!
BLT Ranch Salad Pizza-Pampered Chef from Food.com: Have not tried; posting for future use. Prep time includes the amount of time required for crust to cool.
Maybe with some ranch dressing instead of mayo or dressing mix in the mayo- can be a Bacon Ranch Pizza- My family's favorite from Round Table
I'm on the verge of spontaneous combustion right now. Why yes, I can predict and plan my own spontaneity. That's one of the perks of being a sanguine Leo/Virgo cusp monster. Of course there are other perks, such as eyes in the back of my head (fine, they're cross-eyed) and ninja-like shredding paper skills, but …


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"The X-iT ladder is probably the safest portable fire escape ladder in the world. Its semi-rigid design of inter-locking sections stops I the ladder I from swaying and swinging. Steel rungs have I stand-offs that keep the feet safely off the I wall. Manufactured in the UK from zinc-plated steel, the X-iT is independently tested I to withstand a distributed load of 450kg (992 lbs).The minimum opening width of the window is 300mm (12 inches). Adjustable hooks fit over most walls and can even I be used if there is a radiator I on the inside of the wall. The strong hooks also offer a good handhold. The escape ladder is suitable for walls from 9 inches (23cm) up to 20 inches (51cm) thickness. In an emergency I lift the ladder out of the box, place it over the wall and pull the release toggle, the ladder will then automatically unfold and the sections will inter-lock."
Catalogue Number: 117-9634
- 450
- Steel
- Safe to carry up to 450kg (992lbs)
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Days Inn Toledo Airport
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- Shuttle bus service
- Swimming pool
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- Continental Breakfast
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TripAdvisor Reviews Days Inn Toledo Airport Swanton
Travel Blogs from Swanton
Pretty and warm day
On the road. Blue skies and 90 degrees. Mostly interstate but easy driving. Stopped here to allow a weather system pass over during the night I should make it to Chicago early tomorrow afternoon.
Michigan ~ Lower Peninsula East
... br>
heading on up into Michigan's Upper Peninsula
~ all for now
except i think i have to agree w/ the ...
Turning for home
... goodbyes to Andy and Annett and headed out around noon.. We gave away all our veggies and cutup all our remaining apples for eating while we drove, so we were ready for the border crossing. There were long lineups, but they were mostly all trucks. The car and RV lanes were actually quite short! We waited about 15 minutes and got through in less than 5...no questions about chicken or eggs this time.... The border crossing in Sarnia has been improved and ...
... a few broken spokes and Marie had problems when shifting gears. It was great being able to relax and not bike for a bit.
We also decided to get rid of a lot of things, we were carrying way too much. I'm not sure how much I was carrying but I wasn't able to lift the back of my bicycle, but now I am able ...
The Road Trip begins - more delays!
... we know. The handbrake set up too had me baffled for a while. It is a foot pedal above the left foot rest. Countryside was mainly rural with plenty of healthy looking corn crops spaced between interstate service centres and the exits to major and minor towns such as Toledo, Cleveland among others. Stopped for a Maccas Quarter Pounder at a roadside service centre mid afternoon. Changed drivers to Steve, and continued on until we came to ...


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When Joseph Gordon-Levitt commits to a role, he really goes for it. If you need further proof of that, his role in Jimmy Kimmel's "David After Dentist" adaptation will take care of that.
"Jimmy Kimmel Live's" post-Oscars special took some of the most popular videos on YouTube and turned them into big budget productions. One of those videos was "David After Dentist," though not much of the infamous car video was used for the trailer. Still, that didn't stop Levitt from memorizing every single second of it.
During Thursday's (March 6) episode, Kimmel showed the full take of the scene and says Levitt used no cue cards or prompts. It's pretty shocking how perfectly he has the video down. Please, let this be the video shown with he finally wins an Oscar.


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Karma Points
Study Experts (graduate students and teachers who work for Algebra Nation to moderate) award Karma Points to students for helping other students and starting new discussions about math on the Wall. Students can help other students by directing them to videos that will help them with their questions and by explaining the first step of how to start a particular math problems. But remember, students won't be given Karma Points if they just give the answer to the question another student's question!
Students can also see where they stand by looking at the Karma Points Leaderboard. The number of Karma Points that the students has earned is shown on each wall post they make, on the top of the screen, and on the Leaderboard page.
What do students want to earn Karma Points? The best way to learn math is to help others. Also, in addition to bragging rights, students can win great prizes! Recently, students have won t-shirts, pizza parties, and iPads!
Florida Geometry/Algebra 2 students! Here is what you can win:
From September to April each year, we give out 1 iPads each month to the student who earn the most Karma Points that month. Each month, first place wins an iPad. Second, third, fourth, fifth, and sixth places each win a Math Nation t-shirt.
Please note that while students can win a t-shirt more than 1 time (we have multiple colors now!), students can only win an iPad once.


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Have you used your brain today ?
I don't remember much about it now.
Was I there?
Well I saw you. But I may be conflating that memory with seeing you somewhere else…
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What the heck is a chenopod? That's supposed to be one of our primary
Karen Fernsler wrote:
> Ragweed is not my friend.
> I have been watching this for my area:
> http://www.wunderground.com/DisplayPollen.asp
> Ragweed is not my friend. It tells you what the predominant pollens
> are, but doesn't really break it down.
> Did I mention ? -- ragweed is not my friend.
> Blah.
> On Mon, Aug 21, 2006 at 10:03:34AM -0400, Bonnie & Bill Morgan wrote:
>> Sue, I have terrible allergies, too. Do you know what you are allergic to?
>> What do you do when you have an attack? Is it primarily sinus symptoms? If
>> so, there may be a few things to make you more comfortable, in addition to
>> some of the newer medications (mostly prescription) out on the market. I
>> use Rhinocort with great success. If I'm out, I've found steamy showers,
>> standing over a pot of boiling water or eating a nice large bowl of chicken
>> soup will give some temporary relief. If your DH will give you a nice
>> gentle pressure over your sinus cavities, that too can give temporary
>> relief. There is so much more that can be done to help alleviate symptoms
>> that was not available years ago. And if you get allergy attacks frequently
>> and they keep you from enjoying what you love to do, I'd recommend checking
>> into getting allergy shots. It's a long term investment, but over time, it
>> does seem to help.
>> Here is hoping you are feeling better today!
>> Blessings,
>> Bonnie (SW OH - zone 5)
>> -----Original Message-----
>> From: email@example.com [mailto:firstname.lastname@example.com] On Behalf
>> Of SueNY
>> Sent: Sunday, August 20, 2006 9:28 PM
>> To: firstname.lastname@example.com
>> Subject: Re: [CHAT] Ummm...
>> Haven't done a thing today..not even a visit to the garden. My allergies
>> flared up big time and it feels like I have a stack of bricks on my face.
>> Not fun. :-(
>> Sue(6B)
>> ---------------------------------------------------------------------
>> To sign-off this list, send email to email@example.com with the message
>> text UNSUBSCRIBE GARDENCHAT
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Israel Offers to Help Turkey After Mining Explosion Kills 245Israel offers to send MDA teams to Soma, Turkey, after huge explosion kills hundreds.
Guinea Moves To Bar Israeli Tycoon's Mining RightsAfrican state releases report arguing diamond magnate Steinmetz obtained rights to world's largest iron-ore deposit by corruption.
Israeli Company to Dig for Uranium in the NegevStudies say that there is uranium under the Negev - and Gulliver Energy intends to dig it up.


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Join FREE | Login
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Grands!® Barbecue Chicken Pizzas
Grands!® Pepperoni Pizza Bake
Easy Caprese Pizza Bake
Chicken Alfredo Pizza
Stuffed-Crust Pizza
Deep-dish Pizza Pie Bundle
Enjoy this cheesy barbecue pizza made using Pillsbury® pizza crust and soy burger - a spicy dinner.
Barbecue sauce is sold in many varieties; choose the flavor your family likes best.
One pound of cooked and drained ground beef can be used in place of the soy burgers.
To better distribute the jalapeño chile slices throughout the recipe, halve or quarter larger pieces.
© 2016 ®/TM General Mills All Rights Reserved


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Have you ever purchased from PartSelect.com before?
Thank you for helping make our site better.
Below is a list of all parts for the Maytag Dryer MDE9206AYW - Residential, Electric. To find the repair part you need, you can browse the list below or you can narrow your choices by searching for a part by name or description.
This motor comes with a threaded shaft.
This male door strike is just under an inch long. It is made of metal.
This dryer timer is used to control the unit's cycles by regulating the length of time that power is directed to each setting.
Screws not included.
This screw is sold individually.
Sold individually.
This handy drying rack for inside your dryer is great for items that cannot tumble dry.
Sold Individually.
All brand logos are trademarks of their respective owners.
The PartSelect logo is a Registered Trademark of Atlantic Laundry Centres, Ltd.
Copyright © 1999-2017 , Eldis Group Partnership. All rights reserved.


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Beyond Bioshock: A visual history of Irrational and Turbine
Let the games begin
Quincy's Irrational Games and Needham's Turbine are both major players in the video game industry. Irrational released Bioshock Infinite on March 26 and Turbine has announcing Infinite Crisis, a multiplayer online battle game featuring DC Comics characters. As gamers open their copies of Bioshock Infinite and apply to be beta testers for Infinite Crisis, here's a look back at some of the previous games from these two local companies (along with a quick peek at the new games). Next
Bioshock Infinite
Bioshock Infinite is the highly anticipated (and oft-delayed) follow-up (but not sequel) to Bioshock. The game is set in 1912 and gamers get to play as Booker DeWitt, a man who must pay off his debt by finding a missing girl in a fictional floating city. . Next
Infinite Crisis
Infinite Crisis is a free multiplayer online battle arena featuring characters from the DC Comics universe. The game offers competitive player versus player battle experience set in the DC Multiverse—a world in which familiar DC Comics characters have been reimagined in startling ways. Featuring a wide variety of DC Comics heroes and villains, Infinite Crisis puts players in control of twisted incarnations of their favorite characters throughout the Multiverse such as Nightmare Batman and Gaslight Catwoman, playing in league with well-known characters including Green Lantern and The Flash. Next
Asheron's Call
Launched in 1999, Asheron's Call was the game that put Turbine on the map. A fantasy-adventure game, Asheron's Call lets players customize their alter ego in appearance and balance of heroic skills as they battle in a magical frontier of monsters, breathtaking vistas, and fast alliances. Next
Widely considered one of the best games ever released, Bioshock lets you play as the sole survivor of an oceanic plane crash. According to the Irrational Games website, once your character swims to a lighthouse, you discover a bathysphere and descend to the seafloor where arch-capitalist Andrew Ryan's city sits. Volcanic vents fuel its sprawling Art Deco facilities. Economic liberalism spurred its leading citizens to innovate in the name of prestige and profit; the gene-altering drug ADAM granted them unbelievable abilities and then drove them mad. Saving your skin could cost you your humanity; returning to the surface means delving deeper into the city of Rapture's sordid history. Next
Freedom Force
Released in 2002, Freedom Force is Irrational Games's take on comic books. The game featured superhero characters battling villains and can still be played on PCs through distribution services including Steam. Next
Freedom Force Vs. The Third Reich
Released three years after Freedom Force in 2005, this game had the heroes battling an imagined Third Reich. According to the Irrational Games website, there were also two sequels that were considered, but never made.
"We had actually had two games we almost made," recalls Ken Levine, Creative Director at Irrational Games. "One was set in the Bronze Age of comics – the '70s – and Robb Waters [long time concept artist at Irrational] actually did a huge raft of concepts for it, and then we had a game called "Freedom Force Console" which was going to be a console game, more action oriented, and that was going to be more modern day." Next
Lord of the Rings Online
An online multiplayer universe, Lord of the Rings lets players play characters from the popular books and movies. Players can enter the universe playing a man, Elf, Dwarf, or Hobbit. Next
Tribes: Vengeance
Released in 2004, Tribes: Vengeance is a single-player game that alternates between several characters' points of view. There is also an offline training mode that teaches skills for the first-person shooter's multiplayer modes. Next
Dungeons & Dragons Online
Dungeons & Dragons started as a game played mostly in players' heads, but, Turbine helped translate the experience into a multiplayer online game. The game is truly massive with the company releasing a number of expansions to the online universe. Next
System Shock 2
A first-person shooter mixed with a role-playing game, System Shock 2 offers a sophisticated plot and its fair share of scary moments. Players can choose to specialize in one of three career branches — emphasizing marksmanship, hacking and repair know-how, or paranormal mental powers — and further enhance their skill sets with cyber module upgrades. Back to the beginning


text: Wåven differentiates itself by combining contemporary fashion brand ideologies with Scandinavian design influences at an affordable price. The focus of the product is design and attention to detail. With two generations of denim and manufacturing expertise - fit, fabric, quality and wash are our main priorities and we look to keep true to this. Waven is new to the market and set to take the denim industry by storm with their quality denim and stylish cuts with 70's inspiration. We love Waven and we are sure you will too


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A lovely pendant necklace from Hot Diamonds. Its crafted from sterling silver into a beautiful lucky horseshoe shape, and features a single sparkling diamond. Pendant measures 1.1cm x 1.3cm.
Thank you for visiting Hot Diamonds Hope Sterling Silver Diamond Pendant Necklace, Silver on shoppingfind.co.uk
We bring the products from many websites to one website for you to search, save and compare.


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On Second Thought is a one-hour, daily news talk show that airs at 9 a.m. ET weekdays.
Journey to Palm Springs for vintage and antique finds such as a Roy Lichtenstein screenprint, a NASA archive and a Tiffany Studios mosaic. Monday, February 27 at 8 PM
The GHSA state championships for football are right around the corner. There are 16 teams left on the road to the Georgia Dome and by late Saturday night all eight champions for 2016 will be... MORE
GPB Passport is the new member benefit that provides you with extended access to an on-demand library of quality local and national public television programming, including tens of thousands of hours of your favorite programs.
Mon., July 8, 2013 12:51pm
Personalized Plates Hearing Tuesday
Car owners will get their chance to speak out on the state's newly proposed rules for personalized license plates. On Tuesday, the Department of Revenue will hold a public hearing after a lawsuit forced them to rewrite the regulations. The state is currently operating under emergency regulations for prestige plates. Last month, the state settled with First Amendment attorney Cynthia Counts after officials denied her client three different license plates with the word "gay" in them.
Full Story
Wed., April 18, 2012 9:23am
Pools Scramble To Meet New Rules
Hotels, municipal recreation centers and other swimming pool operators are scrambling to comply with new government regulations requiring public pools to be accessible to people with disabilities.
Sun., February 19, 2012 11:00pm
Bill Would Legalize Living On Boats
A coastal lawmaker says, Georgia is losing business from boaters who want to take up floating residences in the state. Woodbine Republican Jason Spencer introduced a bill in the state legislature to allow live-aboard boats. Georgia outlawed long-term boat stays 20 years ago out of concern for the human waste that some boaters dump in the water.
Wed., January 25, 2012 5:00pm
Lawmakers To Study Small Biz Regulations
State House Speaker David Ralston announced Wednesday that lawmakers will begin accepting feedback about Georgia regulations from small business owners.
Thu., September 15, 2011 12:25pm
Bill Challenges Federal Regulations
Georgia U.S. Senators Johnny Isakson and Saxby Chambliss are among Senate Republicans pushing for a one-year moratorium on new federal regulations. They say the new rules will stifle business. Environmentalists see the moratorium as another example of putting business interests before public health.
Wed., November 11, 2009 3:45am
State Lawmaker Wants Congress to Change Banking Rules
Rep. James Mills says Georgia's banks are failing because federal regulator


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Thanks for helping!
Several hiking trails are located in this area.
We hiked the Hatcher Pass/ Reed Lakes Trail, and let me say, for someone who is not used to hiking this was moderate to difficult hike at times. The first two miles are well paved...
read more
We hiked the Hatcher Pass/ Reed Lakes Trail, and let me say, for someone who is not used to hiking this was moderate to difficult hike at times. The first two miles are well paved and easy hike. Later the trail move into a more rugged trail. There is several boulder fields and that's why this hike becomes more tricky....
I had an afternoon to kill so on the recommendation of locals I drove up to Hatcher Pass, it did not disappoint. Beautiful, rugged mountains with views of the valley below, lots of snow activities available to do up at the top in the winter.
When I was in Alaska in September, my cousin and I drove up to Hatcher Pass on Sept. 3, 2016. The 35 mile drive from the Parks Highway up to Hatcher Pass took about an hour and was bumpy with the road varying between paved to gravel to dirt with potholes. But the amazing views along the way was worth...
Be safe from the snow and bring a truck or don't drive up the road all the way. They usually block it off but this time they didn't and I got stuck in a rental Yaris. Once the road turns to gravel two way traffic is difficult. Fortunately I was saved my 3 amazing men. There is no cell service...
We only had one day in Anchorage before heading to our cruise ship in Seward. Our waitress recommended Hatcher Pass and we are so glad we went! It was about an hour and a half drive up through Wasilla and through the mountains. From the top there were a few different hiking trails you could take to see even more...
Worthy the ride. excellent views all the way from Palmer, the river is beautiful an the views from the Pass are never to be forgotten. The unpaved road was in great conditions.
Hatcher pass has become one of my favorite places to go. It has so many beautiful trails of varying lengths and difficulties. A quick hike to Gold Cord Lake is easy exercise but the views make you feel like you are worlds away from the parking lot below. I also recommend the drive through the pass to willow. It's spectacular....
We stopped here on our way to Independence Mine. We saw it more as a photo op as we didn't have a chance to hike around. On a clear day the views would be spectacular.
We live in Alaska and are used to the beautiful mountains. But there is simply no place like Hatcher's Pass. I lived in Chile, and these mountains are more breathtaking than the Andes.
We took a day drive along Hatcher Pass approaching from Palmer toward the Independence Mine State Historical Park. The road curves along streams and in between mountains for beautiful views. Once past Independence Mine, however, the road turns to gravel and remains that way to Willow, so very slow traveling. But worth it if you have the time. We noticed...
We are going to be there in June with a rental car. How bad is the gravel part of the road and how long is the gravel part. Since it's gravel Google Maps doesn't have it -Thanks A
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Service Oriented Architecture in short SOA is a collection of services used for making communication with each other. The communication can involve either simple data passing or it could involve two or more services coordinating some activities. SOA is a business-centric IT architectural approach that supports integrating your business as linked, repeatable business tasks, or services.
Ask questions pertaining to SOA by posting your query on our new services 'Ask Questions SOA Online'. Here, you can post all your queries relating to SOA including programming problems, subject assignment, uses of SOA, implementation of SOA and project related questions. Our SOA expert will answer all your queries within two days and our solutions will be flawless. We will also get solutions from our visitors as it is allowed for everyone, who knows the answer and post it by logging his/her account. So, feel free and post your questions regarding SOA.
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