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1.  Interferon-alpha competing endogenous RNA network antagonizes microRNA-1270 
Cellular and Molecular Life Sciences  2015;72(14):2749-2761.
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-015-1875-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00018-015-1875-5
PMCID: PMC4477080  PMID: 25746225
Non-coding RNA; Regulatory RNA network; Competing endogenous RNA; Natural antisense RNA; Mechanisms of action
2.  The Anti-Inflammatory Effects of Flavanol-Rich Lychee Fruit Extract in Rat Hepatocytes 
PLoS ONE  2014;9(4):e93818.
Flavanol (flavan-3-ol)-rich lychee fruit extract (FRLFE) is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and the proinflammatory mediator nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS). Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β)-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB). Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α). FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be used to treat inflammatory diseases.
doi:10.1371/journal.pone.0093818
PMCID: PMC3976307  PMID: 24705335
3.  Stabilization of human interferon-α1 mRNA by its antisense RNA 
Cellular and Molecular Life Sciences  2012;70(8):1451-1467.
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-012-1216-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00018-012-1216-x
PMCID: PMC3607724  PMID: 23224365
Interferon-α1 antisense RNA; mRNA stabilization; Interferon-α1 mRNA; microRNA; miR-1270; Regulatory RNA
4.  Peroxidation of n-3 Polyunsaturated Fatty Acids Inhibits the Induction of iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes 
Eicosapentaenoic acid and docosahexaenoic acid (EPA/DHA), n-3 polyunsaturated fatty acids (PUFAs), have a variety of biological activities including anti-inflammatory and anticancer effects. We hypothesized that their peroxidized products contributed in part to anti-inflammatory effects. In the liver, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as one of the factors in hepatic inflammation and injury. We examined whether the peroxidation of EPA/DHA influences the induction of iNOS and NO production in proinflammatory cytokine-stimulated cultured hepatocytes, which is in vitro liver inflammation model. Peroxidized EPA/DHA inhibited the induction of iNOS and NO production in parallel with the increased levels of their peroxidation, whereas unoxidized EPA/DHA had no effects at all. Peroxidized EPA/DHA reduced the activation of transcription factor, NF-κB, and the expression of the iNOS antisense transcript, which are involved in iNOS promoter transactivation (mRNA synthesis) and its mRNA stabilization, respectively. These findings demonstrated that peroxidized products of EPA/DHA suppressed the induction of iNOS gene expression through both of the transcriptional and posttranscriptional steps, leading to the prevention of hepatic inflammation.
doi:10.1155/2011/374542
PMCID: PMC3136170  PMID: 21773019
5.  Acute and late effects on induction of allodynia by acromelic acid, a mushroom poison related structurally to kainic acid 
British Journal of Pharmacology  2004;142(4):679-688.
Ingestion of a poisonous mushroom Clitocybe acromelalga is known to cause severe tactile pain (allodynia) in the extremities for a month and acromelic acid (ACRO), a kainate analogue isolated from the mushroom, produces selective damage of interneurons of the rat lower spinal cord when injected either systemically or intrathecally. Since ACRO has two isomers, ACRO-A and ACRO-B, here we examined their acute and late effects on induction of allodynia.Intrathecal administration of ACRO-A and ACRO-B provoked marked allodynia by the first stimulus 5 min after injection, which lasted over the 50-min experimental period. Dose-dependency of the acute effect of ACRO-A on induction of allodynia showed a bell-shaped pattern from 50 ag kg−1 to 0.5 pg kg−1 and the maximum effect was observed at 50 fg kg−1. On the other hand, ACRO-B induced allodynia in a dose-dependent manner from 50 pg kg−1 to 50 ng kg−1. N-methyl-D-aspartate (NMDA) receptor antagonists and Joro spider toxin, a Ca2+-permeable AMPA receptor antagonist, inhibited the allodynia induced by ACRO-A, but not by ACRO-B. However, other AMPA/kainate antagonists did not affect the allodynia induced by ACRO.Whereas no neuronal damage was observed in the spinal cord in ACRO-A-treated mice, induction of allodynia by ACRO-A (50 fg kg−1) and ACRO-B (50 ng kg−1) was selectively lost 1 week after i.t. injection of a sublethal dose of ACRO-A (50 ng kg−1) or ACRO-B (250 ng kg−1). Higher doses of ACRO-A, however, could evoke allodynia dose-dependently from 50 pg kg−1 to 500 ng kg−1 in the ACRO-A-treated mice. The allodynia induced by ACRO-A (500 ng kg−1) was not inhibited by Joro spider toxin or NMDA receptor antagonists. These properties of the late allodynia induced by ACRO-A were quite similar to those of the acute allodynia induced by ACRO-B.ACRO-A could increase [Ca2+]i in the deeper laminae, rather than in the superficial laminae, of the spinal cord. This increase was not blocked by the AMPA-preferring antagonist GYKI52466 and Joro spider toxin.Taken together, these results demonstrate the stereospecificity of ACRO for the induction of allodynia and suggest the presence of a receptor specific to ACRO.
doi:10.1038/sj.bjp.0705834
PMCID: PMC1575046  PMID: 15159282
Acromelic acid; allodynia; AMPA; kainate; spinal cord
6.  Inhibition of nociceptin-induced allodynia in conscious mice by prostaglandin D2 
British Journal of Pharmacology  1997;122(4):605-610.
We recently showed that intrathecal administration of nociceptin induced allodynia by innocuous tactile stimuli and hyperalgesia by noxious thermal stimuli in conscious mice. In the present study, we examined the effect of prostaglandins on nociceptin-induced allodynia and hyperalgesia.Prostaglandin D2 (PGD2) blocked the allodynia induced by nociceptin in a dose-dependent manner with an IC50 of 26 ng kg−1, but did not affect the nociceptin-induced hyperalgesia at doses up to 500 ng kg−1. BW 245C (an agonist for PGD (DP) receptor) blocked the allodynia with an IC50 of 83 ng kg−1.The blockade of nociceptin-induced allodynia by PGD2 was reversed by the potent and selective DP-receptor antagonist BW A868C in a dose-dependent manner with an ED50 of 42.8 ng kg−1.Glycine (500 ng kg−1) almost completely blocked the nociceptin-induced allodynia. A synergistic effect on the inhibition of nociceptin-evoked allodynia was observed between glycine and PGD2 at below effective doses.Dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, blocked the nociceptin-induced allodynia with an IC50 of 2.9 μg kg−1.PGE2, PGF2α, butaprost (an EP2 agonist) and cicaprost (a PGI receptor agonist) did not affect the nociceptin-induced allodynia.These results demonstrate that PGD2 inhibits the nociceptin-evoked allodynia through DP receptors in the spinal cord and that glycine may be involved in this inhibition.
doi:10.1038/sj.bjp.0701421
PMCID: PMC1564979  PMID: 9375954
Nociceptin; prostaglandin D2; allodynia; hyperalgesia; spinal cord; glycine

Results 1-6 (6)