Widespread 8-oxoguanine modifications of miRNA seeds differentially regulate redox-dependent cancer development.

Oxidative stress contributes to tumourigenesis by altering gene expression. One accompanying modification, 8-oxoguanine (o8G) can change RNA-RNA interactions via o8G•A base pairing, but its regulatory roles remain elusive. Here, on the basis of o8G-induced guanine-to-thymine (o8G > T) variations featured in sequencing, we discovered widespread position-specific o8Gs in tumour microRNAs, preferentially oxidized towards 5' end seed regions (positions 2-8) with clustered sequence patterns and clinically associated with patients in lower-grade gliomas and liver hepatocellular carcinoma. We validated that o8G at position 4 of miR-124 (4o8G-miR-124) and 4o8G-let-7 suppress lower-grade gliomas, whereas 3o8G-miR-122 and 4o8G-let-7 promote malignancy of liver hepatocellular carcinoma by redirecting the target transcriptome to oncogenic regulatory pathways. Stepwise oxidation from tumour-promoting 3o8G-miR-122 to tumour-suppressing 2,3o8G-miR-122 occurs and its specific modulation in mouse liver effectively attenuates diethylnitrosamine-induced hepatocarcinogenesis. These findings provide resources and insights into epitranscriptional o8G regulation of microRNA functions, reprogrammed by redox changes, implicating its control for cancer treatment.

8-Oxoguanine: from oxidative damage to epigenetic and epitranscriptional modification.

In pathophysiology, reactive oxygen species control diverse cellular phenotypes by oxidizing biomolecules. Among these, the guanine base in nucleic acids is the most vulnerable to producing 8-oxoguanine, which can pair with adenine. Because of this feature, 8-oxoguanine in DNA (8-oxo-dG) induces a G > T (C > A) mutation in cancers, which can be deleterious and thus actively repaired by DNA repair pathways. 8-Oxoguanine in RNA (o8G) causes problems in aberrant quality and translational fidelity, thereby it is subjected to the RNA decay pathway. In addition to oxidative damage, 8-oxo-dG serves as an epigenetic modification that affects transcriptional regulatory elements and other epigenetic modifications. With the ability of o8G•A in base pairing, o8G alters structural and functional RNA-RNA interactions, enabling redirection of posttranscriptional regulation. Here, we address the production, regulation, and function of 8-oxo-dG and o8G under oxidative stress. Primarily, we focus on the epigenetic and epitranscriptional roles of 8-oxoguanine, which highlights the significance of oxidative modification in redox-mediated control of gene expression.

AGO-accessible anticancer siRNAs designed with synergistic miRNA-like activity.

Small interfering RNAs (siRNAs) therapeutically induce RNA interference (RNAi) of disease-causing genes, but they also silence hundreds of seed-matched off-targets as behaving similar to microRNAs (miRNAs). miRNAs control the pathophysiology of tumors, wherein their accessible binding sites can be sequenced by Argonaute crosslinking immunoprecipitation (AGO CLIP). Herein, based on AGO CLIP, we develop potent anticancer siRNAs utilizing miRNA-like activity (mi/siRNAs). The mi/siRNAs contain seed sequences (positions 2-7) of tumor-suppressive miRNAs while maintaining perfect sequence complementarity to the AGO-accessible tumor target sites. Initially, host miRNA interactions with human papillomavirus 18 (HPV18) were identified in cervical cancer by AGO CLIP, revealing tumor-suppressive activity of miR-1/206 and miR-218. Based on the AGO-miRNA binding sites, mi/siRNAs were designed to target E6 and E7 (E6/E7) transcript with seed sequences of miR-1/206 (206/E7) and miR-218 (218/E7). Synergistic anticancer activity of 206/E7 and 218/E7 was functionally validated and confirmed via RNA sequencing and in vivo xenograft models (206/E7). Other mi/siRNA sequences were additionally designed for cervical, ovarian, and breast cancer, and available as an online tool (http://ago.korea.ac.kr/misiRNA); some of the mi/siRNAs were validated for their augmented anticancer activity (206/EphA2 and 206/Her2). mi/siRNAs could coordinate miRNA-like activity with robust siRNA function, demonstrating the potential of AGO CLIP analysis for RNAi therapeutics.

Position-specific oxidation of miR-1 encodes cardiac hypertrophy.

In pathophysiology, reactive oxygen species oxidize biomolecules that contribute to disease phenotypes1. One such modification, 8-oxoguanine2 (o8G), is abundant in RNA3 but its epitranscriptional role has not been investigated for microRNAs (miRNAs). Here we specifically sequence oxidized miRNAs in a rat model of the redox-associated condition cardiac hypertrophy4. We find that position-specific o8G modifications are generated in seed regions (positions 2-8) of selective miRNAs, and function to regulate other mRNAs through o8G•A base pairing. o8G is induced predominantly at position 7 of miR-1 (7o8G-miR-1) by treatment with an adrenergic agonist. Introducing 7o8G-miR-1 or 7U-miR-1 (in which G at position 7 is substituted with U) alone is sufficient to cause cardiac hypertrophy in mice, and the mRNA targets of o8G-miR-1 function in affected phenotypes; the specific inhibition of 7o8G-miR-1 in mouse cardiomyocytes was found to attenuate cardiac hypertrophy. o8G-miR-1 is also implicated in patients with cardiomyopathy. Our findings show that the position-specific oxidation of miRNAs could serve as an epitranscriptional mechanism to coordinate pathophysiological redox-mediated gene expression.

siAbasic: a comprehensive database for potent siRNA-6Ø sequences without off-target effects.

Small interfering RNA (siRNA) is widely used to specifically silence target gene expression, but its microRNA (miRNA)-like function inevitably suppresses hundreds of off-targets. Recently, complete elimination of the off-target repression has been achieved by introducing an abasic nucleotide to the pivot (position 6; siRNA-6Ø), of which impaired base pairing destabilizes transitional nucleation (positions 2-6). However, siRNA-6Ø varied in its conservation of on-target activity (∼80-100%), demanding bioinformatics to discover the principles underlying its on-target efficiency. Analyses of miRNA-target interactions (Ago HITS-CLIP) showed that the stability of transitional nucleation correlated with the target affinity of RNA interference. Furthermore, interrogated analyses of siRNA screening efficiency, experimental data and broadly conserved miRNA sequences showed that the free energy of transitional nucleation (positions 2-5) in siRNA-6Ø required the range of stability for effective on-target activity (-6 ≤ ΔG[2:5] ≤ -3.5 kcal mol-1). Taking into consideration of these features together with locations, guanine-cytosine content (GC content), nucleotide stretches, single nucleotide polymorphisms and repetitive elements, we implemented a database named 'siAbasic' that provided the list of potent siRNA-6Ø sequences for most of human and mouse genes (≥ ∼95%), wherein we experimentally validated some of their therapeutic potency. siAbasic will aid to ensure potency of siRNA-6Ø sequences without concerning off-target effects for experimental and clinical purposes.

CLIPick: a sensitive peak caller for expression-based deconvolution of HITS-CLIP signals.

High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP, also called CLIP-Seq) has been used to map global RNA-protein interactions. However, a critical caveat of HITS-CLIP results is that they contain non-linear background noise-different extent of non-specific interactions caused by individual transcript abundance-that has been inconsiderately normalized, resulting in sacrifice of sensitivity. To properly deconvolute RNA-protein interactions, we have implemented CLIPick, a flexible peak calling pipeline for analyzing HITS-CLIP data, which statistically determines the signal-to-noise ratio for each transcript based on the expression-dependent background simulation. Comprising of streamlined Python modules with an easy-to-use standalone graphical user interface, CLIPick robustly identifies significant peaks and quantitatively defines footprint regions within which RNA-protein interactions were occurred. CLIPick outperforms other peak callers in accuracy and sensitivity, selecting the largest number of peaks particularly in lowly expressed transcripts where such marginal signals are hard to discriminate. Specifically, the application of CLIPick to Argonaute (Ago) HITS-CLIP data were sensitive enough to uncover extended features of microRNA target sites, and these sites were experimentally validated. CLIPick enables to resolve critical interactions in a wide spectrum of transcript levels and extends the scope of HITS-CLIP analysis. CLIPick is available at: http://clip.korea.ac.kr/clipick/.

Evaluation and control of miRNA-like off-target repression for RNA interference.

RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago-miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.

MicroRNA Target Recognition: Insights from Transcriptome-Wide Non-Canonical Interactions.

MicroRNAs (miRNAs) are small non-coding RNAs (∼22 nucleotides) regulating gene expression at the post-transcriptional level. By directing the RNA-induced silencing complex (RISC) to bind specific target mRNAs, miRNA can repress target genes and affect various biological phenotypes. Functional miRNA target recognition is known to majorly attribute specificity to consecutive pairing with seed region (position 2-8) of miRNA. Recent advances in a transcriptome-wide method of mapping miRNA binding sites (Ago HITS-CLIP) elucidated that a large portion of miRNA-target interactions in vivo are mediated not only through the canonical "seed sites" but also via non-canonical sites (∼15-80%), setting the stage to expand and determine their properties. Here we focus on recent findings from transcriptome-wide non-canonical miRNA-target interactions, specifically regarding "nucleation bulges" and "seed-like motifs". We also discuss insights from Ago HITS-CLIP data alongside structural and biochemical studies, which highlight putative mechanisms of miRNA target recognition, and the biological significance of these non-canonical sites mediating marginal repression.

Rationally designed siRNAs without miRNA-like off-target repression.

Small interfering RNAs (siRNAs) have been developed to intentionally repress a specific gene expression by directing RNA-induced silencing complex (RISC), mimicking the endogenous gene silencer, microRNAs (miRNAs). Although siRNA is designed to be perfectly complementary to an intended target mRNA, it also suppresses hundreds of off-targets by the way that miRNAs recognize targets. Until now, there is no efficient way to avoid such off-target repression, although the mode of miRNA-like interaction has been proposed. Rationally based on the model called "transitional nucleation" which pre-requires base-pairs from position 2 to the pivot (position 6) with targets, we developed a simple chemical modification which completely eliminates miRNA-like off-target repression (0%), achieved by substituting a nucleotide in pivot with abasic spacers (dSpacer or C3 spacer), which potentially destabilize the transitional nucleation. Furthermore, by alleviating steric hindrance in the complex with Argonaute (Ago), abasic pivot substitution also preserves near-perfect on-target activity (~80-100%). Abasic pivot substitution offers a general means of harnessing target specificity of siRNAs to experimental and clinical applications where misleading and deleterious phenotypes from off-target repression must be considered. [BMB Reports 2016; 49(3): 135-136].

Abasic pivot substitution harnesses target specificity of RNA interference.

Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA-target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼ 80-100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications.

Enhanced attachment of human osteoblasts on NH3-treated poly(L-lactic acid) membranes for guided bone regeneration.

Barrier membranes for guided bone regeneration (GBR) were prepared by a solvent casting method using solutions of poly(L-lactic acid) (PLLA) and chitosan. PLLA and PLLA/chitosan membranes were treated with ammonia gas plasma. PLLA/chitosan membranes were successfully fabricated, and the surface of the PLLA/chitosan membrane was clearly modified by NH3 plasma treatment according to attenuated total reflectance (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) analyses. Additionally, water contact angle testing indicated that the hydrophilicity of these membranes was significantly increased. MG-63 cells were cultured on each type of membrane, and cell viability was examined using an MTT assay. After one week of culturing, MG-63 cells were more abundant on PLLA/chitosan membranes than on PLLA membranes. The cell viability of PLLA/chitosan membranes with plasma treatment was significantly higher than that of PLLA membranes. These results suggest that this plasma-treated membrane is suitable for GBR and is a promising source of bioactive membrane material for bone regeneration.

A comparative study of bone formation following grafting with different ratios of particle dentin and tricalcium phosphate combinations.

This study aimed to evaluate the bone regeneration relative to tooth powder and tricalcium phosphate (TCP) mixing ratios using the rabbit cranium defect model. The tooth powder was mixed with TCP in 1:1, 3:1, and 1:3 ratios, and the different ratios were implanted in the rabbit cranium defect for 4 and 8 weeks. Powders crystal structure evaluated using scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and new bone formation (NBF) was analyzed using micro-computed tomography (CT) and histologic examination. NBF in the control group was restricted to the defect margins. More NBF was observed around the defect margins in the experimental groups compared with the control group. Specifically, active NBF was identified around the implant materials of the centrifugal part of the defect and defect margins in the 3:1 tooth powder: TCP group. Our results suggested that tooth powder and TCP may be useful in bone regeneration.

The influence of personality and dysfunctional sleep-related cognitions on the severity of insomnia.

Previous findings suggest that personality traits and dysfunctional sleep-related cognitions may perpetuate insomnia, but findings concerning this have been scarce. Thus, we hypothesized that personality and sleep-related cognitions influence the severity of insomnia, and investigated the association personality and sleep-related cognitions had with various sleep-related parameters, including severity of insomnia. Forty-four patients with psychophysiological insomnia were assessed using The Temperament and Character Inventory, the Insomnia Severity Index, the Pittsburgh Sleep Quality Index, the Epworth Sleepiness Scale, the Dysfunctional Belief and Attitudes toward Sleep Scale, the Pre-Sleep Arousal Scale and the Hospital Anxiety and Depression Scale. Insomnia severity was significantly and positively correlated with harm avoidance, self-transcendence and sleep-related cognitions, and negatively correlated with novelty seeking, reward dependence, and cooperativeness. Dysfunctional sleep-related cognitions were positively correlated with insomnia severity and sleep quality. Stepwise multiple regression analysis showed that sleep-related cognitions, depression and reward dependence scores were significant determinants of insomnia severity, and that sleep-related cognitions and self-transcendence were significant positive determinants of sleep quality. Reward dependence, depression and sleep-related cognitions were associated with insomnia severity, and comparison with previous findings implied that 'internalizing behavior' and depression may be more plausible candidates for the link between personality and insomnia than anxiety. Considering the major role of cognitive-behavioral treatment (CBT) in the treatment of insomnia, assessment of these factors and management of sleep-related cognitions may help alleviate symptoms in patients with insomnia.

The impact of temperament and character on the efficacy of nonpharmacologic treatment of primary insomnia.

Nonpharmacologic treatment, also known as cognitive behavioral treatment, is a first-line treatment of primary insomnia. We aimed to assess factors, including temperament and character, that were associated with responses to nonpharmacologic treatments of primary insomnia, that may assist physicians to recommend appropriate treatment. Outpatients diagnosed with psychophysiological insomnia (n = 99) were recruited between May 2009 and January 2010. Among 69 patients who consented to participate, 44 completed treatment and all assessment measures. In addition, 37 normal control subjects were also recruited. Baseline characteristics were assessed using the Temperament and Character Inventory, the Insomnia Severity Index, the Pittsburgh Sleep Quality Index, the Epworth Sleepiness Scale, the Dysfunctional Beliefs and Attitudes about Sleep scale, and the Hospital Anxiety and Depression Scale. After treatment, all assessment scales excluding the Temperament and Character Inventory were repeated. All patients received nonpharmacologic treatments, including sleep restriction, cognitive therapy, and sleep hygiene education. Novelty seeking, harm avoidance, reward dependence, cooperativeness, and self-transcendence scores were significantly different between normal controls and study subjects. Participants were divided into treatment responders (n = 23) and nonresponders (n = 21). Responders were significantly younger (50.3 ± 12.8 vs 58.7 ± 9.6 years, P = .02) and had significantly higher reward dependence scores (51.7 ± 5.9 vs 42.9 ± 6.9, P < .01) compared with nonresponders. The difference in reward dependence scores remained significant after controlling for other factors (odds ratio, 1.23; 95% confidence interval, 1.08-1.40; P = .01). Among personality dimensions, reward dependence was significantly associated with response to nonpharmacologic treatment in patients with primary insomnia.

Pax6 expression is sufficient to induce a neurogenic fate in glial progenitors of the neonatal subventricular zone.

BACKGROUND: The forebrain subventricular zone (SVZ) of neonatal mammals contains a large, heterogeneous population of migratory and proliferating precursors of interneurons and glia. These cell types are produced in large numbers in the immediate postnatal period, the glioblasts populating the hemispheres with astrocytes and oligodendrocytes, the neuroblasts migrating to the olfactory bulb to become interneurons. How cell fate decisions are determined or stabilized in this mixed population is not clear, although previous studies indicate the importance of two transcription factors, Pax6 in neurons and Olig2 in glia, and suggest there may be reciprocal repression between these genes. METHODOLOGY/PRINCIPAL FINDINGS: In examining the SVZ of neonatal mouse and rat brain, we find that the very large majority of SVZ cells express either Pax6 or Olig2, but few express both. We have used in vivo retro- and lenti-virus injections into the neonatal SVZ and in vitro gene transfer to demonstrate that pax6 over-expression is sufficient to down-regulate olig2 and to promote a neuronal lineage development and migration pattern in olig2-expressing cells. Furthermore, we provide evidence that Pax6 binds to the olig2 promoter and that an HEB sequence in the promoter is required for the Pax6 repression of olig2 transcription. Lastly, we constructed a lentivirus to target olig2-expressing cells in the SVZ to trace their fates, and found that the very large majority developed into glia. CONCLUSIONS/SIGNIFICANCE: We provide evidence for a direct repression of olig2 by Pax6. Since SVZ cells can display developmental plasticity in vitro, the cross-repression promotes a stabilization of cell fates. This repression may be critical in a germinal zone in which immature cells are highly migratory and are not organized into an epithelium.

Adenosine and purine nucleosides protect rat primary astrocytes from peroxynitrite-potentiated, glucose deprivation-induced death: preservation of intracellular ATP level.

Previously we have reported that immunostimulated astrocytes became highly vulnerable to glucose deprivation. In the present study we examined the effect of various kinds of nucleosides on the augmented death of glucose-deprived immunostimulated astrocytes. Preincubation with interferon-gamma (100 U/ml) and lipopolysaccharide (1 microg/ml) for 48 h and continuous exposure to glucose deprivation (4 h) significantly induced the lactate dehydrogenase (LDH) release, as a marker of cell injury or death, from astrocytes. The glucose deprivation-induced augmented cell death in immunostimulated astrocytes was mimicked by exogenous peroxynitrite generator 3-morpholinosydnonimine (SIN-1). The increased death in immunostimulated or SIN-1-treated astrocytes deprived of glucose was blocked by adenosine and ATP. Other purine nucleos(t)ides, not pyrimidine nucleotides, also showed similar protective effects. Adenosine receptor agonist R(-)-N-(2-phenylisopropyl)-adenosine or N-cyclohexyladenosine did not alter the augmented cell death. Adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener or 3,7-dimethyl-1-propargylxanthine also did not reverse the protective effect of adenosine. Intracellular ATP levels rapidly decreased prior to the LDH release in glucose-deprived immunostimulated astrocytes. The loss of intracellular ATP was prevented by adenosine and other purine nucleotides. The present results suggest that adenosine and their metabolites may protect astrocytes from peroxynitrite-potentiated, glucose deprivation-induced death by serving as substrates for intracellular ATP generation.