Antisense miRNAs Research Articles

Short 2'-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo.

MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.

Abstract 7520: NRT-YHD_001, targeting CD47 for liver cancer immunotherapy

Abstract Previously, we demonstrated that recovery of histone deacetylase 6 (HDAC6) suppressed let-7i-5p expression to de-repress the thrombospondin-1 (TSP1) expression, and thereby it occupied CD47 receptor to block CD47-SIRPα mediated anti-phagocytosis of macrophage in liver cancer. NRT-YHD_001 is a modified antisense miRNA of let-7i-5p for liver cancer treatment. In addition, N-acetylgalactosamine was attached to NRT-YHD_001 for specific delivery to the liver. In vitro phagocytosis assay showed better macrophage phagocytosis activity than non-modified antisense let-7i-5p. To investigate the in vivo efficacy of NRT-YHD_001, spontaneous (Ras-transgenic) mouse liver cancer model was used and compared with that of sorafenib treatment. This mouse liver cancer model usually develops spontaneous liver cancer mass after 15 ~ 20 weeks of birth. We confirmed that both weekly i.v. injection of 1mg/kg and biweekly i.v. injection of 2mg/kg NRT-YHD_001 showed more significant therapeutic efficacy than the sorafenib-treated group. Furthermore, xenograft assay with Hep3B human liver cancer cells in athymic-nude mouse also showed that weekly i.v. injection of 1mg/kg NRT-YHD_001 exhibited better survival and tumor growth inhibition compared to non-treated group. In mouse and cynomolgus monkey maximum tolerated dose (MTD) studies, no toxicity of NRT-YHD_001 was found at doses up to 500 mg/kg intravenously. These results showed that NRT-YHD_001 had a robust anticancer effect by converting the macrophage of “don't eat me” into “eat me signal” in the tumor microenvironment of liver cancer, thereby removing or inhibiting liver cancer cells. Keywords: let-7i-5p, macrophage, phagocytosis, liver cancer, CD47 Citation Format: Suk Woo Nam, Sangyean Kim, Minjeong Na, Eunbi Shin, Jin Woong Ha, Soyoung Jeon, Sungpil Yoon. NRT-YHD_001, targeting CD47 for liver cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7520.

The role of lncRNAKCNQ1OT1/miR-301b/Tcf7 axis in cardiac hypertrophy.

Cardiac hypertrophy, acting as a pathologic process of chronic hypertension and coronary disease, and its underlying mechanisms still need to be explored. Long non-coding RNA (LncRNA) potassium voltage-gated channel subfamily Q member 1 Transcript 1 (KCNQ1OT1) has been implicated in myocardial infarction. However, its role in cardiac hypertrophy remains reported. To explore the regulated effect of lncRNAKCNQ1OT1 and miR-301b in cardiac hypertrophy, gain-and-lose function assays were tested. The expression of lncRNAKCNQ1OT1 and miR-301b were tested by quantitative real time polymerase chain reaction (qRT-PCR). The levels of transcription factor 7 (Tcf7), Proto-oncogene c-myc (c-myc), Brainnatriureticpeptide (BNP)and β-myosin heavy chain (β-MHC) were detected by Western blot. Additionally, luciferase analysis revealed interaction between lncRNAKCNQ1OT1, BNPβ-MHCmiR-301b, and Tcf7. LncRNAKCNQ1OT1 overexpression significantly induced cardiac hypertrophy. Furthermore, lncRNAKCNQ1OT1 acts as a sponge for microRNA-301b, which exhibited lower expression in cardiac hypertrophy model, indicating an anti-hypertrophic role. Furthermore, the BNP and β-MHC expression increased, as well as cardiomyocyte surface area, with Ang II treatment, while the effect was repealed by miR-301b. Moreover, the protein expression of Tcf7 was inversely regulated by miR-301b and Antisense miRNA oligonucleotides (AMO)-301b. Our study has shown that overexpression of lncRNAKCNQ1OT1 could promote the development of cardiac hypertrophy by regulating miR-301b and Tcf7. Therefore, inhibition of lncRNAKCNQ1OT1 might be a potential therapeutic strategy for cardiac hypertrophy.

MicroRNAs and their therapeutic strategy in phase Iand phase IIclinical trials.

miRNAs play a crucial therapeutic role in diseases such as cancer, diabetes and viral infections, with around 1900 identified in the human genome. Some have progressed to clinical trials, and miRNA mimics and miRNA inhibitors are pivotal therapeutic molecules undergoing evaluation. The review delves into various miRNA-associated clinical trials, emphasizing their precision in targeting specific genes, modulating disease pathways and diagnostic potential. This underscores the importance of miRNA therapy, foreseeing innovations in precision medicine techniques for diverse diseases. The future envisions improved delivery systems addressing challenges like immunogenicity and digestion, while a comprehensive miRNA-based omics database could guide the development of tailored antisense miRNAs, further advancing precision medicine strategies.

Next generation sequencing uncovers multiple miRNAs associated molecular targets in gallbladder cancer patients

Gallbladder cancer (GBC) is a lethal disease with surgical resection as the only curative treatment. However, many patients are ineligible for surgery, and current adjuvant treatments exhibit limited effectiveness. Next-generation sequencing has improved our understanding of molecular pathways in cancer, sparking interest in microRNA-based gene regulation. The aim of the study is to identify dysregulated miRNAs in GBC and investigate their potential as therapeutic tools for effective and targeted treatment strategies. GBC and control tissue samples were sequenced for miRNA expression using the Illumina HiSeq platform. Biological processes and related pathways were determined using the Panther and Gene Ontology databases. 439 significantly differentially expressed miRNAs were identified; 19 of them were upregulated and 29 were downregulated. Key enriched biological processes included immune cell apoptosis, endoplasmic reticulum (ER) overload response, and negative regulation of the androgen receptor (AR) signaling pathway. Panther analysis revealed the insulin-like growth factor (IGF)-mitogen activated protein kinases (MAPK) cascade, p38 MAPK pathway, p53 pathway, and FAS (a subgroup of the tumor necrosis factor receptor) signaling pathway as highly enriched among dysregulated miRNAs. Kirsten rat sarcoma virus (KRAS), AR, and interferon gamma (IFN-γ) pathways were identified among the key pathways potentially amenable to targeted therapy. We concluded that a combination approach involving miRNA-based interventions could enhance therapeutic outcomes. Our research emphasizes the importance of precision medicine, targeting pathways using sense and anti-sense miRNAs as potential therapies in GBC.

MiR-31-5p as a Potential Circulating Biomarker and Tracer of Clinical Improvement for Chronic Inflammatory Demyelinating Polyneuropathy.

MicroRNAs are endogenous, small noncoding RNA molecules that play a pivotal role in the regulation of gene expression. MicroRNAs are involved in many biological processes such as proliferation, cell differentiation, neovascularization, and apoptosis. Studies on microRNA expression may contribute to a better understanding of the pathomechanism of chronic inflammatory demyelinating polyneuropathy (CIDP) and consequently enable the development of new therapeutic measures using antisense miRNAs (antagomirs). In this study, we evaluated the level of miR-31-5p in the serum of patients with CIDP and its correlation with the miR-31-5p level and clinical presentation and electrophysiological and biochemical parameters. The study group consisted of 48 patients, mean age 61.60 ± 11.76, who fulfilled the diagnostic criteria of a typical variant of CIDP. The expression of miR-31-5p in patient serum probes was investigated by droplet digital PCR. The results were correlated with neurophysiological findings and the patient's clinical and biochemical parameters. The mean copy number of miRNA-31 in 100 μl serum was 1288.64 ± 2001.02 in the CIDP group of patients, while in the control group, it was 3743.09 ± 4026.90. There was a significant positive correlation (0.426) between IgIV treatment duration and miR-31-5p expression. Patients without IgIV treatment showed significantly lower levels of miR-31 compared to the treated group (259.44 ± 304.02 vs. 1559.48 ± 2168.45; p = 0.002). The group of patients with body weight > 80 kg showed statistically significantly lower levels of miRNA-31-5p than the patients with lower body weight (934.37 ± 1739.66 vs. 1784.62 ± 2271.62, respectively; p = 0.014). Similarly, the patients with elevated cerebrospinal fluid (CSF) protein levels had significantly higher miRNA-31-5p expression than those with normal protein levels (1393.93 ± 1932.27 vs. 987.38 ± 2364.10, respectively; p = 0.044). The results may support the hypothesis that miR-31-5p is strongly involved in the autoimmune process in CIDP. The positive correlation between higher miR-31-5p levels and duration of IVIg treatment may be an additional factor explaining the efficacy of prolonged IVIg therapy in CIDP.

Abstract 5165: In vivo therapeutic validation of NRT-YHD_001, a novel macrophage checkpoint inhibitor, in liver cancer

Abstract Previously, we demonstrated that recovery of Histone deacetylase 6 (HDAC6) elicited let-7i-5p suppression to de-repress the thrombospondin-1 (TSP1) expression, and thereby it occupied CD47 receptor to block CD47-SIRPα mediated anti-phagocytosis of macrophage in liver cancer. NRT-YHD_001 is a modified antisense miRNA of let-7i-5p for liver cancer treatment. NRT-YHD_001 was substituted with O-methoxyethyl and phosphothioate for stability, and N-acetylgalactosamine was attached for specific delivery to the liver. In in vitro phagocytosis assay, NRT-YHD_001 showed macrophage phagocytosis activity with similar or better results compared to non-modified antisense let-7i-5p (AS_let-7i-5p). In addition, it was confirmed that NRT-YHD_001 inhibited endogenous let-7i-5p of liver cancer cells and activated macrophage phagocytosis at 4°C or 25°C for up to 2 weeks. When NRT-YHD_001 activity was investigated in vivo after 1 week, 2 weeks, and 4 weeks after administration of single dose NRT-YHD_001 to the tail vein of a mouse, NRT-YHD_001 was not degraded, and let-7i-5p was inhibited while maintaining activity in liver tissue for 2 weeks after injection. To investigate the in vivo therapeutic effect, a mouse spontaneous (Ras-transgenic) liver cancer model was used and compared with the sorafenib-administered group. After administration of NRT-YHD_001 and sorafenib each week at the time when liver mass was detected, the tumor size and growth rate were compared. As a result, the NRT-YHD_001 administration group showed significant therapeutic effect compared to sorafenib. These results showed that NRT-YHD_001 had a strong anticancer effect by converting the macrophage of don't eat me into eat me signal in the tumor microenvironment of liver cancer, thereby removing or inhibiting liver cancer cells. Keywords: let-7i-5p, macrophage, phagocytosis, liver cancer, NRT-YHD_001 Citation Format: Suk Woo Nam, Sangyean Kim, Min Jeong Na, Eunbi Shin, Jin Woong Ha, Soyoung Jeon, Jeong Hwan Yoon. In vivo therapeutic validation of NRT-YHD_001, a novel macrophage checkpoint inhibitor, in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5165.

Extracellular Vesicles and MicroRNA in Myelodysplastic Syndromes.

The bone marrow niche plays an increasing role in the pathophysiogenesis of myelodysplastic syndromes. More specifically, mesenchymal stromal cells, which can secrete extracellular vesicles and their miRNA contents, modulate the fate of hematopoietic stem cells leading to leukemogenesis. Extracellular vesicles can mediate their miRNA and protein contents between nearby cells but also in the plasma of the patients, being potent tools for diagnosis and prognostic markers in MDS. They can be targeted by antisense miRNA or by modulators of the secretion of extracellular vesicles and could lead to future therapeutic directions in MDS.

The etiological roles of miRNAs, lncRNAs, and circRNAs in neuropathic pain: A narrative review.

BackgroundNon‐coding RNAs (ncRNAs) are involved in neuropathic pain development. Herein, we systematically searched for neuropathic pain‐related ncRNAs expression changes, including microRNAs (miRNAs), long non‐coding RNAs (lncRNAs), and circular non‐coding RNAs (circRNAs).MethodsWe searched two databases, PubMed and GeenMedical, for relevant studies.ResultsPeripheral nerve injury or noxious stimuli can induce extensive changes in the expression of ncRNAs. For example, higher serum miR‐132‐3p, ‐146b‐5p, and ‐384 was observed in neuropathic pain patients. Either sciatic nerve ligation, dorsal root ganglion (DRG) transaction, or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG. lncRNAs, such as early growth response 2‐antisense‐RNA (Egr2‐AS‐RNA) and Kcna2‐AS‐RNA, were upregulated in Schwann cells and inflicted DRG after nerve injury, respectively. Dysregulated circRNA homeodomain‐interacting protein kinase 3 (circHIPK3) in serum and the DRG, abnormally expressed lncRNAs X‐inactive specific transcript (XIST), nuclear enriched abundant transcript 1 (NEAT1), small nucleolar RNA host gene 1 (SNHG1), as well as ciRS‐7, zinc finger protein 609 (cirZNF609), circ_0005075, and circAnks1a in the spinal cord were suggested to participate in neuropathic pain development. Dysregulated miRNAs contribute to neuropathic pain via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–inhibitory imbalances, or exosome miRNA‐mediated neuron–glia communication. In addition, lncRNAs and circRNAs are essential in neuropathic pain by acting as antisense RNA and miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing.ConclusionsNumerous dysregulated ncRNAs have been suggested to participate in neuropathic pain development. However, there is much work to be done before ncRNA‐based analgesics can be clinically used for various reasons such as conservation among species, proper delivery, stability, and off‐target effects.

Rationally identified microRNAs as novel endogenous targets to enhance immunotherapy in glioblastoma multiforme

Glioblastoma Multiforme is the most truculent brain tumor. Current treatment modalities involve chemotherapy, surgery, and radiotherapy. Most GBM patients show chemoresistance over time, and there are not many immunotherapy options available. In cancers, abnormal expression of miRNAs has been associated with tumor initiation, progression, metastasis, and resistance to therapies. Some of the dysregulated miRNAs in cancers can promote tumor growth or act as tumor suppressors. Delivery of miRNA-mimics and anti-sense miRNAs into tumors can directly change the expression of miRNA targets and improve the outcome of GBM therapy.

Posttranscriptional modulation of KCNQ2 gene expression by the miR-106b microRNA family

MicroRNAs (miRNAs) have recently emerged as important regulators of ion channel expression. We show here that select miR-106b family members repress the expression of the KCNQ2 K+ channel protein by binding to the 3'-untranslated region of KCNQ2 messenger RNA. During the first few weeks after birth, the expression of miR-106b family members rapidly decreases, whereas KCNQ2 protein level inversely increases. Overexpression of miR-106b mimics resulted in a reduction in KCNQ2 protein levels. Conversely, KCNQ2 levels were up-regulated in neurons transfected with antisense miRNA inhibitors. By constructing more specific and stable forms of miR-106b controlling systems, we further confirmed that overexpression of precursor-miR-106b-5p led to a decrease in KCNQ current density and an increase in firing frequency of hippocampal neurons, while tough decoy miR-106b-5p dramatically increased current density and decreased neuronal excitability. These results unmask a regulatory mechanism of KCNQ2 channel expression in early postnatal development and hint at a role for miR-106b up-regulation in the pathophysiology of epilepsy.

MicroRNA Delivery by Graphene-Based Complexes into Glioblastoma Cells.

Glioblastoma (GBM) is the most common primary and aggressive tumour in brain cancer. Novel therapies, despite achievements in chemotherapy, radiation and surgical techniques, are needed to improve the treatment of GBM tumours and extend patients’ survival. Gene delivery therapy mostly uses the viral vector, which causes serious adverse events in gene therapy. Graphene-based complexes can reduce the potential side effect of viral carries, with high efficiency of microRNA (miRNA) or antisense miRNA delivery to GBM cells. The objective of this study was to use graphene-based complexes to induce deregulation of miRNA level in GBM cancer cells and to regulate the selected gene expression involved in apoptosis. The complexes were characterised by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy and zeta potential. The efficiency of miRNA delivery to the cancer cells was analysed by flow cytometry. The effect of the anticancer activity of graphene-based complexes functionalised by the miRNA sequence was analysed using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) assays at the gene expression level. The results partly explain the mechanisms of miRNA deregulation stress, which is affected by graphene-based complexes together with the forced transport of mimic miR-124, miR-137 and antisense miR-21, -221 and -222 as an anticancer supportive therapy.

Capturing the RNA castle: Exploiting MicroRNA inhibition for wound healing.

The growing pipelines of RNA-based therapies herald new opportunities to deliver better patient outcomes for complex disorders such as chronic nonhealing wounds associated with diabetes. Members of the microRNA (miRNA) family of small noncoding RNAs have emerged as targets for diverse elements of cutaneous wound repair, and both miRNA enhancement with mimics or inhibition with antisense oligonucleotides represent tractable approaches for miRNA-directed wound healing. In this review, we focus on miRNA inhibition strategies to stimulate skin repair given advances in chemical modifications to enhance the performance of antisense miRNA (anti-miRs). We first explore miRNAs whose inhibition in keratinocytes promotes keratinocyte migration, an essential part of re-epithelialisation during wound repair. We then focus on miRNAs that can be targeted for inhibition in endothelial cells to promote neovascularisation for wound healing in the context of diabetic mouse models. The picture that emerges is that direct comparisons of different anti-miRNAs modifications are required to establish the most translationally viable options in the chronic wound environment, that direct comparisons of the impact of inhibition of different miRNAs are needed to quantify and rank their relative efficacies in promoting wound repair, and that a standardised human ex vivo model of the diabetic wound is needed to reduce reliance on mouse models that do not necessarily enhance mechanistic understanding of miRNA-targeted wound healing.

CRISPR/Cas9 System to Knockdown MicroRNA In Vitro and In Vivo.

MicroRNAs (miRNAs) are a class of small noncoding single-stranded RNA molecules containing 18-22 nucleotides that play an important role in the regulation of gene expression at the post-transcriptional and translational levels. Loss-of-function studies are the fundamental strategy to examine miRNA function and target genes in cellular and molecular biology. Traditional methods for miRNA loss-of-function studies include miRNA-specific antisense inhibitors, miRNA sponges, and genetic knockout. However, efficiency, specificity, and stability of these methods are not adequate. Our study suggests that CRISPR/Cas9 is an economic, convenient, and innovative strategy with high efficiency, specificity, and stability for the modulation of miRNA expression. Herein, we describe a detailed protocol for knocking out miRNA genes in vitro and in vivo with the CRISPR/Cas9 system.

Caspase-Activated Oligonucleotide Probe.

Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resolution. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biological applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.

Articles of Significant Interest in This Issue

During herpes simplex virus 1 (HSV-1) latency, viral gene expression is limited to select noncoding RNAs (ncRNAs), including microRNAs (miRNAs). Barrozo et al. (e00639-20) found that deletion of seed sequences corresponding to the antisense viral miRNAs miR-H1/H6 results in decreased reactivation from explanted murine dorsal root ganglia and latently infected rabbits. The antisense nature of these miRNAs, as well as their differential effects on expression of other ncRNAs in the latent region of the HSV-1 genome, shed light on the complex interactions that regulate the balance between latency and reactivation.

Sense-antisense miRNA pairs constitute an elaborate reciprocal regulatory circuit.

Antisense transcription of protein-coding genes has been increasingly recognized as an important regulatory mechanism of gene expression. However, less is known about the extent and importance of antisense transcription of noncoding genes. Here, we investigate the breadth and dynamics of antisense transcription of miRNAs, a class of important noncoding RNAs. Because the antisense transcript of a miRNA is likely to form a hairpin suitable as the substrate of ADARs, which convert adenosine to inosine in double-stranded RNAs, we used A-to-I RNA editing as ultrasensitive readout for antisense transcription of the miRNAs. Through examining the unstranded targeted RNA-seq libraries covering all miRNA loci in 25 types of human tissues, we identified 7275 editing events located in 81% of the antisense strand of the miRNA loci, thus uncovering the previously unknown prevalent antisense transcription of the miRNAs. We found that antisense transcripts are tightly regulated, and a substantial fraction of miRNAs and their antisense transcripts are coexpressed. Sense miRNAs have been shown to down-regulate the coexpressed antisense transcripts, whereas the act of antisense transcription, rather than the transcripts themselves, regulates the expression of sense miRNAs. RNA editing tends to decrease the miRNA accessibility of the antisense transcripts, therefore protecting them from being degraded by the sense-mature miRNAs. Altogether, our study reveals the landscape of antisense transcription and editing of miRNAs, as well as a previously unknown reciprocal regulatory circuit of sense–antisense miRNA pairs.

Systemic delivery of microRNA-21 antisense oligonucleotides to the brain using T7-peptide decorated exosomes

Antisense miRNA oligonucleotides against miR-21 (AMO-21) have a therapeutic potential for treatment of glioblastoma. However, glioblastoma-targeted delivery through systemic injection requires development of an efficient targeting carrier. For this purpose, a glioblastoma-targeting carrier was developed using the T7 peptide and exosomes. The transferrin receptor is overexpressed on the surface of glioblastoma cells, and T7 is a transferrin receptor-binding peptide. A T7 peptide-decorated exosome (T7-exo) was produced by incorporation of T7 into the exosome membrane as a fusion protein of T7 and Lamp2b. As a control, rabies virus glycoprotein (RVG) peptide targeting brain neuron cells was incorporated into the exosome membrane. AMO-21 was loaded into the exosomes by electroporation. In vitro studies of AMO-21 delivery showed that T7-exo had a higher delivery efficiency to C6 glioblastoma cells than unmodified exosome (Unmod-exo) and RVG-decorated exosome (RVG-exo). For in vivo delivery studies, T7-exo with AMO-21 was delivered into intracranial glioblastoma rat models by intravenous injection through the tail vein. The results showed that T7-exo delivered AMO-21 into the brain more efficiently than Unmod-exo and RVG-exo. In addition, delivery of AMO-21 using T7-exo reduced the miR-21 level in the glioblastoma efficiently. Reduction of miR-21 by AMO-21 induced the expression of PDCD4 and PTEN in tumors, resulting in reduction of tumor sizes. Taken together, these findings indicate that T7-exo is an efficient carrier of AMO-21 into the glioblastoma and may be useful in development of glioblastoma therapy.

Transcriptome-wide identification and characterization of microRNAs responsive to phosphate starvation in Populus tomentosa.

miRNAs (microRNAs) are ~ 21-nt non-coding small RNAs (sRNAs) that play crucial regulatory roles in plant biotic and abiotic stress responses. Phosphorus (Pi) deficiency constrains plant growth and reduces yields worldwide. To identify tree miRNAs and evaluate their functions in the response to low Pi, we identified 261 known and 31 candidate novel miRNA families from three sRNA libraries constructed from Populus tomentosa subjected to sufficient or Pi deficiency condition or to restoration of a sufficient Pi level after Pi deficiency. Pi deficiency resulted in significant changes in the abundance of TPM (transcript per million) of 65 known and 3 novel miRNAs. Interestingly, four miRNAs responsive to low N-miR167, miR394, miR171, and miR857-were found to be involved in the response to low Pi. Thirty-five known and one novel miRNAs responded dynamically to Pi fluctuations, suggesting their involvement in the response to Pi deficiency. miRNA clusters comprising 36 miRNAs were identified in 10 chromosomes. Intriguingly, nine pairs of sense and antisense miRNAs transcribed from the same loci were detected in P. tomentosa, which is the first such report in woody plants. Moreover, target genes of the known miRNAs and novel miRNA candidates with significantly changed abundance were predicted, and their functions were annotated. Degradome sequencing supported the identified targets of miRNAs in P. tomentosa. These findings will enhance our understanding of universal and specific molecular regulatory mechanisms of trees under nutrition stress and may facilitate improvement of the Pi utilization efficiency of woody plants.

Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.

Gammaherpesviruses, including the human pathogens Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, γHV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04-antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04, antisense miRNAs, and the latency gene M2.