Ephrin type-A receptor 2-antisense RNA1/2 promote proliferation and migration of MDA-MB-231 cells through EPHA2-dependent Ras signaling pathway mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 and JUND.

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase that is overexpressed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 expression is regulated. We previously reported that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels and hence production of EPHA2 protein. EPHA2-AS encodes two splice variants, EPHA2-AS1 and EPHA2-AS2. The two variants are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the basal-like/triple-negative molecular subtype of breast cancer cells. In this study, we investigated the mechanism of EPHA2-AS1/2 in triple-negative breast cancer using MDA-MB-231 cells. We performed RNA-seq transcriptome analyses of MDA-MB-231 cells treated with AHCC®, which suppressed expression of EPHA2-AS1/2 and EPHA2 mRNA, and EPHA2-AS1/2-silenced MDA-MB-231 cells. Bioinformatics analyses identified 545 overlapping differentially expressed genes that were significantly up- or down-regulated by these treatments. Subsequent functional enrichment analyses of the overlapping genes in combination with in vitro assays indicated that EPHA2-AS1/2 may promote the proliferation and migration of MDA-MB-231 cells through the EPHA2-dependent Ras signaling pathways mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 (proliferation and migration) and JUND (migration). These results thus suggest that EPHA2-AS1/2 may represent a potential molecular target for triple-negative breast cancer treatment.

EPHA2 antisense RNA modulates EPHA2 mRNA levels in basal-like/triple-negative breast cancer cells.

Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase (RTK), whose over-expression has been observed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 gene (EPHA2) expression is regulated. Here, we report that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels. EPHA2-AS is a ∼1.8 kb long non-coding RNA (lncRNA) with a poly(A) tail that encodes two splice variants, EPHA2-AS1/2. They are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the triple-negative breast cancer (TNBC) subtype. The silencing of EPHA2-AS1/2 by a sense oligonucleotide or over-expression of an antisense oligoribonucleotide, which were both designed from the EPHA2 mRNA region (nt 2955-2974) targeted by AS1/2, showed that EPHA2-AS1/2 modulated EPHA2 mRNA levels by interacting with the specific AS1/2-complementary region in the mRNA. The EPHA2-AS1/2 did not prevent microRNAs from acting on the relevant microRNA response elements shared by EPHA2-AS1/2 and EPHA2 mRNA. Our studies demonstrate a crucial role played by EPHA2-AS1/2 in modulating EPHA2 mRNA levels, and hence production of EPHA2 protein, a key oncogenic RTK that contributes to the tumorigenesis of TNBC cells.

[Non-coding Natural Antisense RNA: Mechanisms of Action in the Regulation of Target Gene Expression and Its Clinical Implications].

Recent advances in high-throughput technologies have revealed that 75% of the human genome is transcribed to RNA, whereas only 3% of transcripts are translated into proteins. Consequently, many long non-coding RNAs (lncRNAs) have been identified, which has improved our understanding of the complexity of biological processes. LncRNAs comprise multiple classes of RNA transcripts that regulate the transcription, stability and translation of protein-coding genes in a genome. Natural antisense transcripts (NATs) form one such class, and the GENCODE v30 catalog contains 16193 lncRNA loci, of which 5611 are antisense loci. This review outlines our emerging understanding of lncRNAs, with a particular focus on how lncRNAs regulate gene expression using interferon-α1 (IFN-α1) mRNA and its antisense partner IFN-α1 antisense (as)RNA as an example. We have identified and characterized the asRNA that determines post-transcriptional IFN-α1 mRNA levels. IFN-α1 asRNA stabilizes IFN-α1 mRNA by cytoplasmic sense-antisense duplex formation, which may enhance the accessibility of an RNA stabilizer protein or decrease the affinity of an RNA decay factor for the RNA. IFN-α1 asRNA can also act as competing molecules in the competing endogenous (ce)RNA network with other members of the IFNA multigene family mRNAs/asRNAs, and other cellular mRNA transcripts. Furthermore, antisense oligoribonucleotides representing functional domains of IFN-α1 asRNA inhibit influenza virus proliferation in the respiratory tract of virus-infected animals. Thus, these findings support, at least in part, the rationale that dissecting the activity of NAT on gene expression regulation promises to reveal previously unanticipated biology, with potential to provide new therapeutic approaches to diseases.

Regulatory non-coding RNAs in nervous system development and disease.

Recent evidence demonstrates that long non-coding RNAs (lncRNAs) regulate the expression of multiple genes in an epigenetic, transcriptional, or post-transcriptional manner. They are involved in various cellular phenomena, such as the recruitment of transcription factors, epigenetic chaperoning, control of alternative splicing, mRNA stability and translational activity, as well as acting as decoys against microRNAs. In this review, we summarize the pivotal roles of lncRNAs in regulation of the gene expression involved in neural cell differentiation, synaptogenesis and synaptic plasticity in the central nervous system (CNS). We also describe the aberrant expression of multiple lncRNAs involved in the pathogenesis of neurological diseases. The abnormal expression of lncRNAs leads to altered expression levels of target genes, which contributes to neurodegenerative diseases, such as in Alzheimer's disease and Parkinson's disease, and to the formation of tumors, such as glioma. Accordingly, we discuss recent findings for the modes of action of lncRNAs in normal CNS development and for aberrant lncRNA actions in the pathogenesis of neuronal diseases.

A guinea pig IFNA1 gene with antiviral activity against human influenza virus infection.

We previously reported a natural antisense (AS) RNA as an important modulator of human interferon-Alpha1 (IFNA1) mRNA levels. Here, we identified the guinea pig (Cavia porcellus) IFNA1 gene to enable a proof-of-concept experiment to be performed to confirm that the AS-mRNA regulatory axis exerts in vivo control over innate immunity. We selected a guinea pig model system for influenza virus infection because guinea pigs encode a functional Mx1 gene, an important anti-viral effector in the type I interferon pathway. We identified 15 guinea pig IFNA1 gene candidates upon bioinformatic analysis and selected the three candidates with the highest sequence homology to Homo sapiens, Mus musculus and Marmota himalayana IFNA1. The anti-viral activity of guinea pig IFN-Alpha1 protein against influenza virus A/Puerto Rico/8/34- or endomyocarditis virus-infection was then determined for the three gene candidates. We identified cpIFNA1 as the candidate with the highest sequence homologies and best anti-viral effects. cpIFNA1 will enable us to perform a proof-of-concept experiment to verify that IFN-Alpha1 AS increases cpIFNA1 mRNA levels, resulting in inhibition of influenza virus proliferation in vivo.

IFN-Alpha1 antisense RNA represses human influenza A virus growth in a guinea pig system.

We reported a natural antisense (AS) long non-coding RNA as an important modulator of interferon-Alpha1 (IFNA1) mRNA levels. We showed that IFN-Alpha1 AS promotes IFNA1 mRNA stability by transient duplex formation and inhibition of miR-1270-induced mRNA decay. Here, we performed a proof-of-concept experiment to verify that the AS-mRNA regulatory axis exerts in vivo control of innate immunity. We established a model system for influenza virus infection using guinea pig, which encodes a functional MX1 gene for the type I IFN pathway. This system allowed us to investigate the effects of antisense oligoribonucleotides representing functional domains of guinea pig IFN-Alpha1 AS on gpIFNA1 mRNA levels and, consequently, on viral proliferation in the respiratory tract of influenza virus-infected animals. We demonstrated that pulmonary-administered asORNs inhibited the proliferation of the virus in the animals by modulating IFNA1 mRNA levels. These results indicate that, in light of the proposed actions, asORNs may modulate the level of IFNA1 mRNA in vivo, indicating that IFN-Alpha1 AS plays a pivotal role in determining the outcome of type I IFN responses.

Pathogen-associated regulatory non-coding RNAs and oncogenesis.

Among all new cancer cases in 2012, on average, 15.4% were caused by Helicobacter pylori or oncoviruses, including Epstein-Barr virus, human papillomavirus, hepatitis B virus, hepatitis C viruses, Kaposi sarcoma-associated herpesvirus and human T-lymphotropic virus. These pathogens encode a variety of non-coding RNAs, which are important cofactors for oncogenesis. In this review, we focus on recent developments in the study of long and small non-protein-coding RNAs, including microRNAs, of oncogenic pathogens, and discuss their mechanisms of action in the multiple steps of oncogenesis.

Interferon-alpha competing endogenous RNA network antagonizes microRNA-1270.

A new form of circuitry for gene regulation has been identified in which RNAs can crosstalk by competing for shared microRNAs (miRNAs). Such competing endogenous RNAs (ceRNAs) form a network via shared miRNA response elements (MREs) to antagonize miRNA function. We previously reported natural antisense RNA (AS) as an important modulator of interferon-α1 (IFN-α1) mRNA levels by promoting IFN-α1 mRNA stability. We show that IFN-α1 AS forms a ceRNA network with specific IFN-α AS (IFN-α7/-α8/-α10/-α14) and mRNA (IFN-α8/-α10/-α14/-α17) subtypes from the IFN-α gene (IFNA) family to antagonize miRNA-1270 (miR-1270), thereby modulating IFN-α1 mRNA levels. Bioinformatic analysis demonstrated that IFN-α1 AS harbors multiple miR-1270 MREs (MRE-1270s), whose presence was substantiated by miR-1270 overexpression and transfection of antimiR-1270. The antimiR-1270, complementary to the miR-1270 seed region, revealed that IFN-α1 AS likely shares the MRE-1270 with IFN-α1 mRNA and specific IFN-α AS and mRNA subtypes. Subsequent bioinformatic analysis for MRE-1270s showed that IFN-α1 AS and other RNA subtypes shared the 6-mer MRE-1270 site. Further MRE-mapping demonstrated that the total number of MRE-1270s in IFN-α1 AS accounted for approximately 30 % of the miR-1270 population. AntimiR-1270 transfection also caused specific de-repression of five cellular mRNAs, including that of CAPRIN1. These results suggest that IFN-α1 AS, together with specific IFN-α AS and mRNA subtypes, as well as the five cellular mRNAs, participate as competing molecules in the ceRNA network against miR-1270. This coordinated regulatory architecture suggests a vital function for the innate immune system in maintaining precise physiological type I IFN levels via post-transcriptional regulatory mechanisms.

Post-transcriptional inducible gene regulation by natural antisense RNA.

Accumulating data indicate the existence of natural antisense transcripts (asRNAs), frequently transcribed from eukaryotic genes and do not encode proteins in many cases. However, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. A low-copy-number asRNA may post-transcriptionally regulate gene expression with cis-controlling elements on the mRNA. The asRNA itself may act as regulatory RNA in concert with trans-acting factors, including various RNA-binding proteins that bind to cis-controlling elements, microRNAs, and drugs. A novel mechanism that regulates mRNA stability includes the interaction of asRNA with mRNA by hybridization to loops in secondary structures. Furthermore, recent studies have shown that the functional network of mRNAs, asRNAs, and microRNAs finely tunes the levels of mRNA expression. The post-transcriptional mechanisms via these RNA-RNA interactions may play pivotal roles to regulate inducible gene expression and present the possibility of the involvement of asRNAs in various diseases.

Interleukin-1ß induces tumor necrosis factor-α secretion from rat hepatocytes.

AIM: Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF-α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS). METHODS: Primary cultured rat hepatocytes were used to analyze TNF-α expression in response to the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). Livers of rats subjected to LPS-induced endotoxemia were analyzed. RESULTS: Immunocytochemistry and enzyme-linked immunosorbent assays demonstrated that IL-1ß-treated rat hepatocytes secreted TNF-α, and RNA analyses indicated that TNF-α mRNA was induced specifically by IL-1ß. Northern blot analysis showed that not only mRNA, but also a natural antisense transcript (asRNA), was transcribed from the rat Tnf gene in IL-1ß-treated hepatocytes. TNF-α was detected in the hepatocytes of LPS-treated rats. Both TNF-α mRNA and asRNA were expressed in the hepatocytes of LPS-treated rats, human hepatocellular carcinoma and human monocyte/macrophage cells. To disrupt the interaction between TNF-α asRNA and TNF-α mRNA, sense oligonucleotides corresponding to TNF-α mRNA were introduced into rat hepatocytes resulting in significantly increased levels of TNF-α mRNA. One of these sense oligonucleotides increased a half-life of TNF-α mRNA, suggesting that the TNF-α asRNA may reduce the stability of TNF-α mRNA. CONCLUSION: IL-1ß-stimulated rat hepatocytes are a newly identified source of TNF-α in the liver. TNF-α mRNA and asRNA are expressed in rats and humans, and the TNF-α asRNA reduces the stability of the TNF-α mRNA. Hepatocytes and TNF-α asRNA may be therapeutic targets to regulate levels of TNF-α mRNA.

Stabilization of human interferon-α1 mRNA by its antisense RNA.

Antisense transcription is a widespread phenomenon in the mammalian genome and is believed to play a role in regulating gene expression. However, the exact functional significance of antisense transcription is largely unknown. Here, we show that natural antisense (AS) RNA is an important modulator of interferon-α1 (IFN-α1) mRNA levels. A ~4-kb, spliced IFN-α1 AS RNA targets a single-stranded region within a conserved secondary structure element of the IFN-α1 mRNA, an element which was previously reported to function as the nuclear export element. Following infection of human Namalwa lymphocytes with Sendai virus or infection of guinea pig 104C1 fetal fibroblasts with influenza virus A/PR/8/34, expression of IFN-α1 AS RNA becomes elevated. This elevated expression results in increased IFN-α1 mRNA stability because of the cytoplasmic (but not nuclear) interaction of the AS RNA with the mRNA at the single-stranded region. This results in increased IFN-α protein production. The silencing of IFN-α1 AS RNA by sense oligonucleotides or over-expression of antisense oligoribonucleotides, which were both designed from the target region, confirmed the critical role of the AS RNA in the post-transcriptional regulation of IFN-α1 mRNA levels. This AS RNA stabilization effect is caused by the prevention of the microRNA (miRNA)-induced destabilization of IFN-α1 mRNA due to masking of the miR-1270 binding site. This discovery not only reveals a regulatory pathway for controlling IFN-α1 gene expression during the host innate immune response against virus infection but also suggests a reason for the large number of overlapping complementary transcripts with previously unknown function.

Regulation of inducible gene expression by natural antisense transcripts.

Natural antisense transcripts are frequently transcribed from many genes in eukaryotes. Although natural antisense transcripts have been recognized for a long time, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various external stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. Recent studies have shed light on the functions of natural antisense transcripts at the post-transcriptional level. An antisense transcript may regulate gene expression with cis-controlling elements on the mRNA, and the antisense transcript itself may act in concert with trans-acting factors, including various proteins that bind to cis-controlling elements, drugs, and microRNAs. A novel mechanism recently reported to regulate mRNA stability includes the interaction of the antisense transcript with mRNA by hybridization to single-stranded loops in secondary structures. This antisense transcript-mediated post-transcriptional regulation may be one of the general mechanisms for the regulation of inducible gene expression and presents the possibility of the involvement of natural antisense transcripts in disease.

Nuclear and cytoplasmic effects of human CRM1 on HIV-1 production in rat cells.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, mediates the nuclear export of unspliced gag and singly spliced env mRNAs by bridging viral RNA and the export receptor, CRM1. Recently, rat CRM1 was found to be less efficient than human CRM1 in supporting Rev function in rats. In this study, to understand the role of CRM1 in HIV propagation, the mechanism underlying the function of human and rat CRM1 in HIV-1 replication was investigated in rat cells. The production of viral particles, represented by the p24 Gag protein, was greatly enhanced by hCRM1 expression in rat cells; however, this effect was not simply because of the enhanced export of gag mRNA. The translation initiation rate of gag mRNA was not increased, nor was the Gag protein stabilized in the presence of hCRM1. However, the processing of the p55 Gag precursor and the release of viral particles were facilitated. These results indicated that hCRM1 exports gag mRNA to the cytoplasm, not only more efficiently than rCRM1 but also correctly, leading to efficient processing of Gag proteins and particle formation.

Novel cis-active structures in the coding region mediate CRM1-dependent nuclear export of IFN-α 1 mRNA.

We recently reported the chromosome region maintenance 1 (CRM1)-dependent nuclear export of intron-less human interferon-α1 (IFN-α1) mRNA, which encodes a main effecter of host innate immunity. We show that the coding region of IFN-α1 mRNA forms novel secondary structures that are responsible for the CRM1-dependent export of the transcript. Deletion-mutagenesis, in vivo export assays, and computer analyses of the folding potentials of export-competent fragments revealed the presence of a domain, termed the conserved secondary structure (CSS), comprising two adjacent putative stable stem-loop structures (nt 208-452). Internal deletion-mutagenesis and constitutive export assays of each stem-loop structure demonstrated that subregions 308-322 and 352-434 act as a core element by conferring the export function on the CSS. Leptomycin B (LMB) inhibition of the CRM1 pathway decreased the export of core element RNA, implying that the principal site of CRM1 action for exporting IFN-α1 mRNA resides within the core element. An RNPS1 (RNA-binding protein S1, serine-rich domain) cDNA was isolated by yeast three-hybrid screening, using bait containing two CSS regions. We showed that RNPS1 might recognize IFN-α1 mRNP that includes CRM1. The data demonstrate that interaction between RNA structures in the coding region and CRM1 affects the nucleocytoplasmic translocation of IFN-α1 mRNA.

Bioisostere of valtrate, anti-HIV principle by inhibition for nuclear export of Rev.

Rational design by the MO calculation disclosed 5,6-dihydrovaltrate (2) as the bioisostere of valtrate (1), the Rev-export inhibitor with anti-HIV activity. The synthesis of 2 was accomplished by ingenious use of asymmetric Diels-Alder reaction and stereoselective epoxidation associated with the adjacent hydroxyl group. Because of similar biological potency to 1, the analog 2 should be recognized as a promising scaffold for new anti-HIV agents with an unprecedented mechanism of action, inhibition for nuclear export of Rev protein, in the conventional remedy.

Halogenated analogs of 1'-acetoxychavicol acetate, Rev-export inhibitor from Alpinia galanga, designed from mechanism of action.

In the course of search for the robust analogs of 1'-acetoxychavicol acetate (ACA, 1), the Rev-export inhibitor from the medicinal plant Alpinia galanga, we clarified formation of the quinone methide intermediate ii to be essential for exerting the inhibitory activity of 1. Based on this mechanism of action, the rational design from the MO calculation of the conclusive activation energy to ii resulted in the four halogenated analogs with more potent activity than ACA (1). In particular, the difluoroanalog 20d exhibited approximately four-fold potent activity as compared with 1.

Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

New Rev-export inhibitor from Alpinia galanga and structure-activity relationship.

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, an HIV-1 viral regulatory protein, disclosed 1'-acetoxychavicol acetate (ACA, 1) as a new inhibitor for nuclear export of Rev from the roots of Alpinia galanga. Both analysis for mechanism of action with biotinylated probe (2) and several synthesized analogs established crucial portions in 1 for Rev-export inhibitory activity.

Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes.

UNLABELLED: During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1beta (IL-1beta) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3'-untranslated region (3'UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5-kilobase region 3'-flanking to iNOS gene exon 27 was involved in IL-1beta induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3'UTR. These results together with the data in a yeast RNA-hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU-rich element-binding protein HuR, a human homolog of embryonic lethal-abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA-binding proteins may mutually interact. CONCLUSION: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA-RNA interaction may be a new therapeutic target for NO-mediating inflammatory diseases.