Expression Of Natural Antisense Transcripts Research Articles

Global identification of natural antisense transcripts in Gossypium hirsutum and Gossypium barbadense under chilling stress

Natural antisense transcripts (NATs) in model plants have been recognized as important regulators of gene expression under abiotic stresses. However, the functional roles of NATs in crops under low temperature are still unclear. Here, we identified 815 and 689 NATs from leaves of Gossypium hirsutum and G.barbadense under chilling stress. Among those, 224 NATs were identified as interspecific homologs between the two species. The correlation coefficients for expression of NATs and their cognate sense genes (CSG) were 0.43 and 0.37 in G.hirsutum and G.barbadense, respectively. Furthermore, expression of interspecific NATs and CSGs alike was highly consistent under chilling stress with correlation coefficients of 0.90-0.91. Four cold-associated NATs were selected for functional validation using virus-induced gene silencing (VIGS). Our results suggest that CAN1 engage in the molecular regulation of chilling stress by regulating SnRK2.8 expression. This highly conserved NAT have valuable potential for applications in breeding cold-tolerant cotton.

Identification cloning and functional analysis of novel natural antisense lncRNA CFL1-AS1 in cattle

ABSTRACT Long noncoding RNAs have been identified as important regulators of gene expression and animal development. The expression of natural antisense transcripts (NATs) transcribed in the opposite direction to protein-coding genes is usually positively correlated with the expression of homologous sense genes and is the key factor for expression. Here, we identified a conserved noncoding antisense transcript, CFL1-AS1, that plays an important role in muscle growth and development. CFL1-AS1 overexpression and knockout vectors were constructed and transfected into 293T and C2C12 cells. CFL1-AS1 positively regulated CFL1 gene expression, and the expression of CFL2 was also downregulated when CFL1-AS1 was knocked down. CFL1-AS1 promoted cell proliferation, inhibited apoptosis and participated in autophagy. This study expands the research on NATs in cattle and lays a foundation for the study of the biological function of bovine CFL1 and its natural antisense chain transcript CFL1-AS1 in bovine skeletal muscle development. The discovery of this NAT can provide a reference for subsequent genetic breeding and data on the characteristics and functional mechanisms of NATs.

Natural antisense transcript of MYOG regulates development and regeneration in skeletal muscle by shielding the binding sites of MicroRNAs of MYOG mRNA 3′UTR

Natural antisense transcripts (NATs) are endogenous RNAs opposite to sense transcripts, and they can significantly contribute to regulating various biological processes through multiple epigenetic mechanisms. NATs can affect their sense transcripts to regulate the growth and development of skeletal muscle. Our analysis of third-generation full-length transcriptome sequencing data revealed that NATs represented a significant portion of the lncRNA, accounting for up to 30.19%–33.35%. The expression of NATs correlated with myoblast differentiation, and genes expressing NATs were mainly involved in RNA synthesis, protein transport, and cell cycle. We found a NAT of MYOG (MYOG-NAT) in the data. We found that the MYOG-NAT could promote the differentiation of myoblasts in vitro. Additionally, knockdown of MYOG-NAT in vivo led to muscle fiber atrophy and muscle regeneration retardation. Molecular biology experiments demonstrated that MYOG-NAT enhances the stability of MYOG mRNA by competing with miR-128-2-5p, miR-19a-5p, and miR-19b-5p for binding to MYOG mRNA 3′UTR. These findings suggest that MYOG-NAT plays a critical role in skeletal muscle development and provides insights into the post-transcriptional regulation of NATs.

Tissue-specific transcriptomic analysis uncovers potential roles of natural antisense transcripts in Arabidopsis heat stress response.

Natural antisense transcripts (NATs) are an important class of non-coding ribonucleic acids (RNAs) that have been shown to regulate gene expression. Using strand-specific RNA sequencing, 36,317 NAT pairs were identified, and 5,536 were specifically expressed under heat stress. We found distinct expression patterns between vegetative and reproductive tissues for both coding genes and genes encoding NATs. Genes for heat-responsive NATs are associated with relatively high levels of H3K4me3 and low levels of H3K27me2/3. On the other hand, small RNAs are significantly enriched in sequence overlapping regions of NAT pairs, and a large number of heat-responsive NATs pairs serve as potential precursors of nat-siRNAs. Collectively, our results suggest epigenetic modifications and small RNAs play important roles in the regulation of NAT expression, and highlight the potential significance of heat-inducible NATs.

PTB-AS, a Novel Natural Antisense Transcript, Promotes Glioma Progression by Improving PTBP1 mRNA Stability with SND1.

Glioma, the most common primary malignancy in the brain, has high recurrence and lethality rates, and thus, elucidation of the molecular mechanisms of this incurable disease is urgently needed. Poly-pyrimidine tract binding protein (PTBP1, also known as hnRNP I), an RNA-binding protein, has various mechanisms to promote gliomagenesis. However, the mechanisms regulating PTBP1 expression are unclear. Herein, we report a novel natural antisense noncoding RNA, PTB-AS, whose expression correlated positively with PTBP1 mRNA. We found that PTB-AS significantly promoted the proliferation and migration invivo and invitro of glioma cells. PTB-AS substantially increased the PTBP1 level by directly binding to its 3' UTR and stabilizing the mRNA. Furthermore, staphylococcal nuclease domain-containing 1 (SND1) dramatically increased the binding capacity between PTB-AS and PTBP1 mRNA. Mechanistically, PTB-AS could mask the binding site of miR-9 in the PTBP1-3' UTR; miR-9 negatively regulates PTBP1. To summarize, we revealed that PTB-AS, which maintains the PTBP1 level through extended base pairing to the PTBP1 3' UTR with the assistance of SND1, could significantly promote gliomagenesis.

Global identification of Arabidopsis lncRNAs reveals the regulation of MAF4 by a natural antisense RNA

Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and plant development. Here, we identified 6,510 lncRNAs in Arabidopsis under normal or stress conditions. We found that the expression of natural antisense transcripts (NATs) that are transcribed in the opposite direction of protein-coding genes often positively correlates with and is required for the expression of their cognate sense genes. We further characterized MAS, a NAT-lncRNA produced from the MADS AFFECTING FLOWERING4 (MAF4) locus. MAS is induced by cold and indispensable for the activation of MAF4 transcription and suppression of precocious flowering. MAS activates MAF4 by interacting with WDR5a, one core component of the COMPASS-like complexes, and recruiting WDR5a to MAF4 to enhance histone 3 lysine 4 trimethylation (H3K4me3). Our study greatly extends the repertoire of lncRNAs in Arabidopsis and reveals a role for NAT-lncRNAs in regulating gene expression in vernalization response and likely in other biological processes.

The temporal expression profile of a Nos3-related natural antisense RNA in the brain suggests a possible role in neurogenesis

Experimental work over the past several years has revealed an unexpected abundance of long natural antisense transcripts (NATs) in eukaryotic species. In light of the proposed role of such RNA molecules in the regulation of gene expression in the brain, attention is now focused on specific examples of neuronal NATs. Of particular interest are NATs that are complementary to mRNAs encoding nitric oxide synthase (NOS), the enzyme responsible for production of the important gaseous neurotransmitter nitric oxide (NO). Here we study the temporal expression profile of murine Nos3as NAT in the brain. Notably, Nos3as NAT is known to act as a negative regulator of Nos3 gene expression. The results of our quantitative analysis reveal differential expression of Nos3as NAT during embryonic and post-embryonic stages of development of the brain. Also, they show that the low levels of Nos3as NAT coincides with active neurogenesis. In addition we report on an inverse correlation between the relative expression level of Nos3as NAT and the level of Nos3 protein. Thus our data raise the hypothesis that the Nos3as NAT regulates neurogenesis through suppression of Nos3 gene activity. This idea is further supported by experiments conducted on the olfactory bulbs and cultured neuroblastoma cells.

Natural antisense transcripts are significantly involved in regulation of drought stress in maize.

Natural antisense transcripts (NATs) are a prominent and complex class of regulatory RNAs. Using strand-specific RNA sequencing, we identified 1769 sense and antisense transcript pairs (NAT pairs) in two maize inbreds with different sensitivity to drought, as well as in two derivative recombination inbred lines (RILs). A significantly higher proportion of NATs relative to non-NATs are specifically expressed under water stress (WS). Surprisingly, expression of sense and antisense transcripts produced by NAT pairs is significantly correlated, particularly under WS. We found an unexpected large proportion of NATs with protein coding potential, as estimated by ribosome release scores. Small RNAs significantly accumulate within NAT pairs, with 21 nt smRNA particularly enriched in overlapping regions of these pairs of genes. The abundance of these smRNAs is significantly altered in the leafbladeless1 mutant, suggesting that these genes may be regulated by the tasiRNA pathway. Further, NATs are significantly hypomethylated and include fewer transposable element sequences relative to non-NAT genes. NAT gene regions also exhibit higher levels of H3K36me3, H3K9ac, and H3K4me3, but lower levels of H3K27me3, indicating that NAT gene pairs generally exhibit an open chromatin configuration. Finally, NAT pairs in 368 diverse maize inbreds and 19 segregating populations were specifically enriched for polymorphisms associated with drought tolerance. Taken together, the data highlight the potential impact of that small RNAs and histone modifications have in regulation of NAT expression, and the significance of NATs in response to WS.

Derivation of an endogenous small RNA from double-stranded Sox4 sense and natural antisense transcripts in the mouse brain

Natural antisense transcripts (NATs) are involved in cellular development and regulatory processes. Multiple NATs at the Sox4 gene locus are spatiotemporally regulated throughout murine cerebral corticogenesis. In the study, we evaluated the potential functional role of Sox4 NATs at Sox4 gene locus. We demonstrated Sox4 sense and NATs formed dsRNA aggregates in the cytoplasm of brain cells. Over expression of Sox4 NATs in NIH/3T3 cells generally did not alter the level of Sox4 mRNA expression or protein translation. Upregulation of a Sox4 NAT known as Sox4ot1 led to the production of a novel small RNA, Sox4_sir3. Its biogenesis is Dicer1-dependent and has characteristics resemble piRNA. Expression of Sox4_sir3 was observed in the marginal and germinative zones of the developing and postnatal brains suggesting a potential role in regulating neurogenesis. We proposed that Sox4 sense-NATs serve as Dicer1-dependent templates to produce a novel endo-siRNA- or piRNA-like Sox4_sir3.

Exploring Differentially Expressed Genes and Natural Antisense Transcripts in Sheep (Ovis aries) Skin with Different Wool Fiber Diameters by Digital Gene Expression Profiling.

Wool fiber diameter (WFD) is the most important economic trait of wool. However, the genes specifically controlling WFD remain elusive. In this study, the expression profiles of skin from two groups of Gansu Alpine merino sheep with different WFD (a super-fine wool group [FD = 18.0 ± 0.5 μm, n= 3] and a fine wool group [FD=23.0±0.5μm, n=3]) were analyzed using next-generation sequencing–based digital gene expression profiling. A total of 40 significant differentially expressed genes (DEGs) were detected, including 9 up-regulated genes and 31 down-regulated genes. Further expression profile analysis of natural antisense transcripts (NATs) showed that more than 30% of the genes presented in sheep skin expression profiles had NATs. A total of 7 NATs with significant differential expression were detected, and all were down-regulated. Among of 40 DEGs, 3 DEGs (AQP8, Bos d2, and SPRR) had significant NATs which were all significantly down-regulated in the super-fine wool group. In total of DEGs and NATs were summarized as 3 main GO categories and 38 subcategories. Among the molecular functions, cellular components and biological processes categories, binding, cell part and metabolic process were the most dominant subcategories, respectively. However, no significant enrichment of GO terms was found (corrected P-value >0.05). The pathways that were significantly enriched with significant DEGs and NATs were mainly the lipoic acid metabolism, bile secretion, salivary secretion and ribosome and phenylalanine metabolism pathways (P < 0.05). The results indicated that expression of NATs and gene transcripts were correlated, suggesting a role in gene regulation. The discovery of these DEGs and NATs could facilitate enhanced selection for super-fine wool sheep through gene-assisted selection or targeted gene manipulation in the future.

Differential expression of natural antisense transcripts during liver development in embryonic mice.

The mature adult liver is a metabolizing and synthesizing organ consisting of hepatic lobules. The liver development during fetal stages is induced by the interaction between hepatoblast cells derived from the endoderm and stellate cells and/or endothelial cells derived from the mesoderm. Recently, the association of non-coding RNAs, such as microRNAs, in the liver development process has been reported. In the present study, the gene expression changes were investigated of natural antisense transcripts (NATs), a type of non-coding RNA, during the liver development using livers from embryonic day (E) 14, E17, E19 and newborn (NB). The upregulated NATs of 87 genes and the downregulated NATs of 26 genes were identified when the expression was >5.0-fold up/downregulated NATs at E17, E19 or NB compared to those at E14, as examined by microarray analysis. Among these NATs, strand-specific reverse transcription-quantitative polymerase chain reaction analyses validated that the NAT expression of Fga, Fgb, Fgg, F2, Apoa1, Fabp1, C3 and Proc, which have metabolic and synthesizing functions in the adult liver, increased, whereas the NAT expression of Ermap and Tfrc, which function in the hematopoiesis, decreased during the developmental stages. These results indicate that the differential expression of several NATs is induced during the process of liver development, suggesting that these NATs may be involved in the regulation of gene expression and functional conversion from a hematopoietic to a metabolic organ in the developmental stages of the liver.

Expression of non-protein-coding antisense RNAs in genomic regions related to autism spectrum disorders.

BackgroundAutism spectrum disorders (ASD) manifest with neurodevelopmental phenotypes including communicative, social and behavioral impairments that affect as many as 1 in 88 children. The majority of autism cases have no known genetic cause, suggesting complex genetics of the disorder, but a few genes of large effect have been identified.MethodsIn order to identify novel ASD genetic correlates, we investigated non-protein coding RNAs (ncRNAs) which are abundantly transcribed from the human genome, enriched in the brain, and have been implicated in neurodevelopmental disorders. Using an algorithm that we developed, we examined a publicly available transcriptomics database, AceView, to identify the natural antisense transcripts (NATs) that overlap with known autism-related genes. We validated the presence and differential expression of NATs in different brain regions of ASD and control brains using qRT-PCR. Additionally, we investigated the subcellular localization of these transcripts in a neuronal cell line using RNA-sequencing (RNA-seq).ResultsWe found noncoding antisense RNA transcripts at approximately 40% of loci previously implicated in ASD. We confirmed the expression of 10 antisense RNAs in different postmortem human brain tissues. The expression of five antisense transcripts was found to be region-specific, suggesting a role for these ncRNAs in the development and function of specific brain regions. Some antisense RNAs overlapping suspected ASD genes exhibited concordant expression relative to their sense protein-coding genes, while other sense-antisense pairs demonstrate a discordant relationship. Interestingly, the antisense RNA corresponding to the SYNGAP1 locus (SYNGAP1-AS) was found to be differentially expressed in brain regions of patients with ASD compared to control individuals. RNA-seq analysis of subcellular compartments from SH-SY5Y human neuroblastoma cells demonstrated that antisense RNAs to ASD candidate genes are predominantly expressed in the nucleoplasmic or chromatin compartments, implying their involvement in nuclear-associated processes.ConclusionsOur data suggests that NATs are abundantly expressed from ASD-related loci and provide evidence for their roles in target gene regulation, neurodevelopment and autism pathogenesis. This class of RNA should therefore be considered in functional studies aimed at understanding genetic risk factors for ASD.

Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

The functions of natural antisense transcripts

NATs (natural antisense transcripts) are widespread in eukaryotic genomes. Experimental evidence indicates that sense and antisense transcripts interact, suggesting a role for NATs in the regulation of gene expression. On the other hand, the transcription of a gene locus in both orientations and RNA hybrid formation can also lead to transcriptional interference, trigger an immune response or induce gene silencing. Tissue-specific expression of NATs and the compartmentalization of cells ensure that the regulatory impact of NATs prevails. Consequently, NATs are now acknowledged as important modulators of gene expression. New mechanisms of action and important biological roles of NATs keep emerging, making regulatory RNAs an exciting and quickly moving area of research.

Conserved expression of natural antisense transcripts in mammals

BackgroundRecent studies had found thousands of natural antisense transcripts originating from the same genomic loci of protein coding genes but from the opposite strand. It is unclear whether the majority of antisense transcripts are functional or merely transcriptional noise.ResultsUsing the Affymetrix Exon array with a modified cDNA synthesis protocol that enables genome-wide detection of antisense transcription, we conducted large-scale expression analysis of antisense transcripts in nine corresponding tissues from human, mouse and rat. We detected thousands of antisense transcripts, some of which show tissue-specific expression that could be subjected to further study for their potential function in the corresponding tissues/organs. The expression patterns of many antisense transcripts are conserved across species, suggesting selective pressure on these transcripts. When compared to protein-coding genes, antisense transcripts show a lesser degree of expression conservation. We also found a positive correlation between the sense and antisense expression across tissues.ConclusionOur results suggest that natural antisense transcripts are subjected to selective pressure but to a lesser degree compared to sense transcripts in mammals.

Natural antisense transcripts in fungi

Fungi are models for investigating many eukaryotic molecular processes. The identification of natural antisense transcripts (NATs) in fungi led to the discovery of mechanisms for controlling gene expression through transcriptional interference, chromatin remodelling and dsRNA formation. An overview of these mechanisms and the description of specific NAT functions is provided to give context for a broader discussion of fungal NATs. Transcriptome analyses have revealed a large number of NATs in a divergent group of fungi. The timing of NAT expression suggests roles in core life functions, such as responding to the environment and sexual reproduction. The transcriptome studies also uncover a large number of NATs whose functions remain elusive. These could provide novel control of gene expression, targeted responses to stimuli, or other functions. The goal of this review is provide background for this expanding field of research while highlighting opportunities for future discoveries.

Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.

Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring

BackgroundThe Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam.ResultsA normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring.ConclusionsThe transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.

993 INFLUENCE OF CHEMOKINE RANTES PROMOTER DIMORPHISMS ON HEPATOCELLULAR CARCINOMA OCCURRENCE IN PATIENTS WITH CIRRHOSIS

detection of new biomarkers for Hepatocellular carcinoma (HCC) is highly needed. We focused on Natural antisense transcripts (NATs), which constitute a class of non-coding RNAs that emerged as important regulators of gene expression. We analyzed NATs expression of HCC to find a novel molecular marker using custom microarray. Methods: 29 patients with HCC were investigated; Hepatitis B virus (HBV) and Hepatitis C virus (HCV)-free HCC (8 cases), HBVassociated HCC (4 cases), HCV-associated HCC (16 cases), and HBV and HCV-associated HCC (1 case). Total RNAs isolated from HCC tissues and corresponding non-cancerous liver tissues from surgically resected samples were subjected to expression analysis using a custom-microarray containing human sense/antisense probes for ca. 21,000 genes. Examination items of sense/antisense RNA were as follows i. HCC and non-cancerous liver tissue, and ii. viral infection of non-cancerous liver tissue. Results: i. 864 RNA including 285 NATs were identified as upand downregulated in HCC tissues, compared to non-cancerous liver tissue (P 2.0). Hierarchical clustering of antisense RNA clearly distinguished HCC and normal liver tissue with or without viral infection. ii. We analyzed the influence of viral infection at non-cancerous liver tissue, 316 RNA including 169 NATs were identified as upand down-regulated between historical background of viral infection (P < 0.001). Cluster analysis revealed that NATs expression was significantly correlated with the viral infection of HBV or HCV. Conclusions: Our study suggests that NATs expression of HCC is significantly different from normal liver tissue. Expression profiling of NATs is effective techniques for diagnosis of HCC carcinogenesis. In addition, analysis of the obtained NATs is helpful to understand the molecular changes in HCC progression and may lead to the identification of new targets for HCC diagnosis.

Identification of natural antisense transcripts involved in human colorectal cancer development

Natural antisense transcripts (NATs) constitute a class of non-coding RNAs that have emerged as important regulators of gene expression. However, involvement of NATs in colorectal cancer (CRC) development has not been reported to date. In the present study, the up- and down-regulation of NATs were investigated in human CRC for their possible involvement in CRC development. Total RNAs isolated from 51 CRC tissues, 9 corresponding non-cancerous tissues and 19 liver metastatic tissues from surgically resected samples were subjected to expression analysis using a custom-microarray containing human sense/antisense probes for ca. 21,000 genes. Comparing CRC tissues with non-cancerous tissues, we identified 415 NATs differentially expressed in CRC and non-cancerous tissues to a significant degree (p<0.001, fold change >4.0 or ≤4.0). When a hierarchical clustering was performed on CRC and non-cancerous samples using these 415 NATs, the samples were separately clustered. Principal component analysis with the same NATs showed clear separation of CRC and non-cancerous samples using the first two principal components (PC1, 80%; PC2, 10%). To validate the expression results obtained from the microarray, the expressions of the 3 selected NATs were examined by strand-specific RT-qPCR, revealing that these expression profiles were consistent with those obtained from microarray analysis. In addition, the NAT expression patterns were found to be different between primary tumors with liver metastasis and those without liver metastasis. In conclusion, these findings taken together indicated that NATs identified in the present study would be involved in CRC development as well as possibly in its metastasis.