Characterization of a miRNA Signature with Enhanced Diagnostic and Prognostic Power for Patients with Bladder Carcinoma.

Bladder carcinoma is globally among the most prevalent cancers and is associated with a high mortality rate at advanced stages. Its detection relies on invasive diagnostic methods that are unpleasant for the patient. Non-invasive molecular biomarkers, such as miRNAs, could serve as alternatives for early detection and prognosis of this malignancy. We designed a computational approach that combines transcriptome profiling, survival analyses, and calculation of diagnostic power in order to isolate miRNA signatures with high diagnostic and prognostic utility. Our analysis of TCGA-BLCA data from 429 patients yielded one miRNA signature, consisting of five upregulated and three downregulated miRNAs with cumulative diagnostic power that outperforms current diagnostic methods. The same miRNAs have a strong prognostic significance since their expression is associated with the overall survival of bladder cancer patients. We evaluated the expression of this signature in 19 solid cancer types, supporting its unique diagnostic utility for bladder carcinoma. We provide computational evidence regarding the functional implications of this miRNA signature in cell cycle regulation, demonstrating its abundance in body fluids, including peripheral blood and urine. Our study characterized a novel miRNA signature with the potential to serve as a non-invasive method for bladder cancer diagnosis and prognosis.

Transcriptome and proteome analysis reveals the anti-cancer properties of Hypnea musciformis marine macroalga extract in liver and intestinal cancer cells.

BACKGROUND: Marine seaweeds are considered as a rich source of health-promoting compounds by the food and pharmaceutical industry. Hypnea musciformis is a marine red macroalga (seaweed) that is widely distributed throughout the world, including the Mediterranean Sea. It is known to contain various bioactive compounds, including sulfated polysaccharides, flavonoids, and phlorotannins. Recent studies have investigated the potential anticancer effects of extracts from H. musciformis demonstrating their cytotoxic effects on various cancer cell lines. The anticancer effects of these extracts are thought to be due to the presence of bioactive compounds, particularly sulfated polysaccharides, which have been shown to have anticancer and immunomodulatory effects. However, further studies are needed to fully understand the molecular mechanisms that underlie their anticancer effects and to determine their potential as therapeutic agents for cancer treatment. METHODS: H. musciformis was collected from the Aegean Sea (Greece) and used for extract preparation. Transcriptome and proteome analysis was performed in liver and colon cancer human cell lines following treatment with H. musciformis seaweed extracts to characterize its anticancer effect in detail at the molecular level and to link transcriptome and proteome responses to the observed phenotypes in cancer cells. RESULTS: We have identified that treatment with the seaweed extract triggers a p53-mediated response at the transcriptional and protein level in liver cancer cells, in contrast to colon cancer cells in which the effects are more associated with metabolic changes. Furthermore, we show that in treated HepG2 liver cancer cells, p53 interacts with the chromatin of several target genes and facilitates their upregulation possibly through the recruitment of the p300 co-activator. CONCLUSIONS: Overall, the available evidence suggests that extracts from H. musciformis have the potential to serve as a source of anticancer agents in liver cancer cells mainly through activation of a p53-mediated anti-tumor response that is linked to inhibition of cellular proliferation and induction of cell death.

Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus.

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations.

A species-specific lncRNA modulates the reproductive ability of the asian tiger mosquito.

Long non-coding RNA (lncRNA) research has emerged as an independent scientific field in recent years. Despite their association with critical cellular and metabolic processes in plenty of organisms, lncRNAs are still a largely unexplored area in mosquito research. We propose that they could serve as exceptional tools for pest management due to unique features they possess. These include low inter-species sequence conservation and high tissue specificity. In the present study, we investigated the role of ovary-specific lncRNAs in the reproductive ability of the Asian tiger mosquito, Aedes albopictus. Through the analysis of transcriptomic data, we identified several lncRNAs that were differentially expressed upon blood feeding; we called these genes Norma (NOn-coding RNA in Mosquito ovAries). We observed that silencing some of these Normas resulted in significant impact on mosquito fecundity and fertility. We further focused on Norma3 whose silencing resulted in 43% oviposition reduction, in smaller ovaries and 53% hatching reduction of the laid eggs, compared to anti-GFP controls. Moreover, a significant downregulation of 2 mucins withing a neighboring (∼100 Kb) mucin cluster was observed in smaller anti-Norma3 ovaries, indicating a potential mechanism of in-cis regulation between Norma3 and the mucins. Our work constitutes the first experimental proof-of-evidence connecting lncRNAs with mosquito reproduction and opens a novel path for pest management.

Antioxidant and Anticancer Activities of Synthesized Methylated and Acetylated Derivatives of Natural Bromophenols.

Natural bromophenols are important secondary metabolites in marine algae. Derivatives of these bromophenol are potential candidates for the drug development due to their biological activities, such as antioxidant, anticancer, anti-diabetic and anti-inflammatory activity. In our present study, we have designed and synthesized a series of new methylated and acetylated bromophenol derivatives from easily available materials using simple operation procedures and evaluated their antioxidant and anticancer activities on the cellular level. The results showed that 2.,3-dibromo-1-(((2-bromo-4,5-dimethoxybenzyl)oxy)methyl)-4,5-dimethoxybenzene (3b-9) and (oxybis(methylene))bis(4-bromo-6-methoxy-3,1-phenylene) diacetate (4b-3) compounds ameliorated H2O2-induced oxidative damage and ROS generation in HaCaT keratinocytes. Compounds 2.,3-dibromo-1-(((2-bromo-4,5-dimethoxybenzyl)oxy)methyl)-4,5-dimethoxybenzene (3b-9) and (oxybis(methylene) )bis(4-bromo-6-methoxy-3,1-phenylene) diacetate (4b-3) also increased the TrxR1 and HO-1 expression while not affecting Nrf2 expression in HaCaT. In addition, compounds (oxybis(methylene)bis(2-bromo-6-methoxy-4,1-phenylene) diacetate (4b-4) inhibited the viability and induced apoptosis of leukemia K562 cells while not affecting the cell cycle distribution. The present work indicated that some of these bromophenol derivatives possess significant antioxidant and anticancer potential, which merits further investigation.

ERK signaling controls productive HIF-1 binding to chromatin and cancer cell adaptation to hypoxia through HIF-1α interaction with NPM1.

The hypoxia-inducible factor HIF-1 is essential for oxygen homeostasis. Despite its well-understood oxygen-dependent expression, regulation of its transcriptional activity remains unclear. We show that phosphorylation by extracellular signal-regulated kinases1/2 (ERK1/2), in addition to promoting HIF-1α nuclear accumulation, also enhances its interaction with chromatin and stimulates direct binding to nucleophosmin (NPM1), a histone chaperone and chromatin remodeler. NPM1 is required for phosphorylation-dependent recruitment of HIF-1 to hypoxia response elements, its interaction with acetylated histones, and high expression of HIF-1 target genes under hypoxia. Transcriptome analysis revealed a significant number of hypoxia-related genes commonly regulated by NPM1 and HIF-1. These NPM1/HIF-1α co-upregulated genes are enriched in three different cancer types, and their expression correlates with hypoxic tumor status and worse patient prognosis. In concert, silencing of NPM1 expression or disruption of its association with HIF-1α inhibits metabolic adaptation of cancer cells and triggers apoptotic death upon hypoxia. We suggest that ERK-mediated phosphorylation of HIF-1α regulates its physical interaction with NPM1, which is essential for the productive association of HIF-1 with hypoxia target genes and their optimal transcriptional activation, required for survival under low oxygen or tumor growth.

Non-Coding Variants in Cancer: Mechanistic Insights and Clinical Potential for Personalized Medicine.

The cancer genome is characterized by extensive variability, in the form of Single Nucleotide Polymorphisms (SNPs) or structural variations such as Copy Number Alterations (CNAs) across wider genomic areas. At the molecular level, most SNPs and/or CNAs reside in non-coding sequences, ultimately affecting the regulation of oncogenes and/or tumor-suppressors in a cancer-specific manner. Notably, inherited non-coding variants can predispose for cancer decades prior to disease onset. Furthermore, accumulation of additional non-coding driver mutations during progression of the disease, gives rise to genomic instability, acting as the driving force of neoplastic development and malignant evolution. Therefore, detection and characterization of such mutations can improve risk assessment for healthy carriers and expand the diagnostic and therapeutic toolbox for the patient. This review focuses on functional variants that reside in transcribed or not transcribed non-coding regions of the cancer genome and presents a collection of appropriate state-of-the-art methodologies to study them.

Targeting Smyd3 by next-generation antisense oligonucleotides suppresses liver tumor growth.

The oncogenic function of suppressor of variegation, enhancer of zeste and MYeloid-Nervy-DEAF1-domain family methyltransferase Smyd3 has been implicated in various malignancies, including hepatocellular carcinoma (HCC). Here, we show that targeting Smyd3 by next-generation antisense oligonucleotides (Smyd3-ASO) is an efficient approach to modulate its mRNA levels in vivo and to halt the growth of already initiated liver tumors. Smyd3-ASO treatment dramatically decreased tumor burden in a mouse model of chemically induced HCC and negatively affected the growth rates, migration, oncosphere formation, and xenograft growth capacity of a panel of human hepatic cancer cell lines. Smyd3-ASOs prevented the activation of oncofetal genes and the development of cancer-specific gene expression program. The results point to a mechanism by which Smyd3-ASO treatment blocks cellular de-differentiation, a hallmark feature of HCC development, and, as a result, it inhibits the expansion of hepatic cancer stem cells, a population that has been presumed to resist chemotherapy.

Nanopore long-read RNA-seq and absolute quantification delineate transcription dynamics in early embryo development of an insect pest.

The olive fruit fly, Bactrocera oleae, is the most important pest for the olive fruit but lacks adequate transcriptomic characterization that could aid in molecular control approaches. We apply nanopore long-read RNA-seq with internal RNA standards allowing absolute transcript quantification to analyze transcription dynamics during early embryo development for the first time in this organism. Sequencing on the MinION platform generated over 31 million reads. Over 50% of the expressed genes had at least one read covering its entire length validating our full-length approach. We generated a de novo transcriptome assembly and identified 1768 new genes and a total of 79,810 isoforms; a fourfold increase in transcriptome diversity compared to the current NCBI predicted transcriptome. Absolute transcript quantification per embryo allowed an insight into the dramatic re-organization of maternal transcripts. We further identified Zelda as a possible regulator of early zygotic genome activation in B. oleae and provide further insights into the maternal-to-zygotic transition. These data show the utility of long-read RNA in improving characterization of non-model organisms that lack a fully annotated genome, provide potential targets for sterile insect technic approaches, and provide the first insight into the transcriptome landscape of the developing olive fruit fly embryo.

LncRNAs as Chromatin Regulators in Cancer: From Molecular Function to Clinical Potential.

During the last decade, high-throughput sequencing efforts in the fields of transcriptomics and epigenomics have shed light on the noncoding part of the transcriptome and its potential role in human disease. Regulatory noncoding RNAs are broadly divided into short and long noncoding transcripts. The latter, also known as lncRNAs, are defined as transcripts longer than 200 nucleotides with low or no protein-coding potential. LncRNAs form a diverse group of transcripts that regulate vital cellular functions through interactions with proteins, chromatin, and even RNA itself. Notably, an important regulatory aspect of these RNA species is their association with the epigenetic machinery and the recruitment of its regulatory apparatus to specific loci, resulting in DNA methylation and/or post-translational modifications of histones. Such epigenetic modifications play a pivotal role in maintaining the active or inactive transcriptional state of chromatin and are crucial regulators of normal cellular development and tissue-specific gene expression. Evidently, aberrant expression of lncRNAs that interact with epigenetic modifiers can cause severe epigenetic disruption and is thus is closely associated with altered gene function, cellular dysregulation, and malignant transformation. Here, we survey the latest breakthroughs concerning the role of lncRNAs interacting with the epigenetic machinery in various forms of cancer.

Cucumber mosaic virus Isolates from Greek Legumes are Associated with Satellite RNAs that are Necrogenic for Tomato.

Worldwide, Cucumber mosaic virus (CMV) is the causal agent of many economically important diseases. Based on immunological or molecular analysis, three distinct subgroups of CMV isolates can be identified (IA, IB, and II). In addition, some CMV isolates are associated with satellite RNAs (satRNAs), a type of noncoding transcript that may alter the symptoms of CMV infections. This study presents an analysis of CMV isolates occurring in legumes in Greece in respect to their genetic diversity, and the presence and diversity of their satRNA. Phylogenetic analysis of the CMV coat protein sequence of 18 legume and 5 tomato CMV isolates collected throughout Greece classified them within subgroups IA and IB, with a limited genetic diversity. The CMV satRNAs found in nine field legumes exhibiting mild symptoms and in one tomato with a necrotic syndrome contained a functional necrogenic motif; therefore, they were grouped within the necrogenic group of CMV-satRNAs. The necrotic phenotype was expressed in all legume CMV isolates containing necrogenic satRNAs when mechanically inoculated onto tomato plants. To our knowledge, this is the first observation that legumes host necrogenic CMV-satRNAs. The possible role of legumes in the epidemiology of CMV and necrogenic satRNA complex is discussed.

Crosstalk mechanisms between the WNT signaling pathway and long non-coding RNAs.

The WNT/ß-catenin signaling pathway controls a plethora of biological processes throughout animal development and adult life. Because of its fundamental role during animal lifespan, the WNT pathway is subject to strict positive and negative multi-layered regulation, while its aberrant activity causes a wide range of pathologies, including cancer. At present, despite the inroads into the molecules involved in WNT-mediated transcriptional responses, the fine-tuning of WNT pathway activity and the totality of its target genes have not been fully elucidated. Over the past few years, long non-coding RNAs (lncRNAs), RNA transcripts longer that 200nt that do not code for proteins, have emerged as significant transcriptional regulators. Recent studies show that lncRNAs can modulate WNT pathway outcome by affecting gene expression through diversified mechanisms, from the transcriptional to post-translational level. In this review, we selectively discuss those lncRNA-mediated mechanisms we believe the most important to WNT pathway modulation.

Smyd3-associated regulatory pathways in cancer.

SMYD3 is a member of the SET and MYND-domain family of methyl-transferases, the increased expression of which correlates with poor prognosis in various types of cancer. In liver and colon tumors, SMYD3 is localized in the nucleus, where it interacts with RNA Pol II and H3K4me3 and functions as a selective transcriptional amplifier of oncogenes and genes that control cell proliferation and metastatic spread. Smyd3 expression has a high discriminative power for the characterization of liver tumors and positively correlates with poor prognosis. In lung and pancreatic cancer, SMYD3 acts in the cytoplasm, potentiating oncogenic Ras/ERK signaling through the methylation of the MAP3K2 kinase and the subsequent release from its inhibitor. A clinico-pathological analysis of lung cancer patients uncovers prognostic significance of SMYD3 only for first progression survival. However, stratification of patients according to their smoking history significantly expands the prognostic value of SMYD3 to overall survival and other features, suggesting that smoking-related effects saturate the clinical analysis and mask the function of SMYD3 as an oncogenic potentiator.

A Positive Regulatory Loop between a Wnt-Regulated Non-coding RNA and ASCL2 Controls Intestinal Stem Cell Fate.

The canonical Wnt pathway plays a central role in stem cell maintenance, differentiation, and proliferation in the intestinal epithelium. Constitutive, aberrant activity of the TCF4/ß-catenin transcriptional complex is the primary transforming factor in colorectal cancer. We identify a nuclear long non-coding RNA, termed WiNTRLINC1, as a direct target of TCF4/ß-catenin in colorectal cancer cells. WiNTRLINC1 positively regulates the expression of its genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/ß-catenin to mediate the juxtaposition of its promoter with the regulatory regions of ASCL2. ASCL2, in turn, regulates WiNTRLINC1 transcriptionally, closing a feedforward regulatory loop that controls stem cell-related gene expression. This regulatory circuitry is highly amplified in colorectal cancer and correlates with increased metastatic potential and decreased patient survival. Our results uncover the interplay between non-coding RNA-mediated regulation and Wnt signaling and point to the diagnostic and therapeutic potential of WiNTRLINC1.

Smyd3 Is a Transcriptional Potentiator of Multiple Cancer-Promoting Genes and Required for Liver and Colon Cancer Development.

Smyd3 is a protein methyltransferase implicated in cancer development. Here we show that Smyd3 expression in mice is required for chemically induced liver and colon cancer formation. In these organs Smyd3 functions in the nucleus, stimulating the transcription of several key regulators involved in cell proliferation, epithelial-mesenchymal transition, the JAK/Stat3 oncogenic pathway, as well as the Myc and Ctnnb1 oncogenes. Smyd3 interacts with H3K4Me3-modified histone tails, which facilitates its recruitment to the core promoter regions of most active genes. Smyd3 binding density on target genes positively correlates with increased RNA polymerase-II density and transcriptional outputs. Despite its widespread distribution, the transcription-potentiating function of Smyd3 is restricted to a particular set of genes, whose expression is induced specifically during carcinogenesis.

Natural diversity in daily rhythms of gene expression contributes to phenotypic variation.

Daily rhythms of gene expression provide a benefit to most organisms by ensuring that biological processes are activated at the optimal time of day. Although temporal patterns of expression control plant traits of agricultural importance, how natural genetic variation modifies these patterns during the day and how precisely these patterns influence phenotypes is poorly understood. The circadian clock regulates the timing of gene expression, and natural variation in circadian rhythms has been described, but circadian rhythms are measured in artificial continuous conditions that do not reflect the complexity of biologically relevant day/night cycles. By studying transcriptional rhythms of the evening-expressed gene gigantea (GI) at high temporal resolution and during day/night cycles, we show that natural variation in the timing of GI expression occurs mostly under long days in 77 Arabidopsis accessions. This variation is explained by natural alleles that alter light sensitivity of GI, specifically in the evening, and that act at least partly independent of circadian rhythms. Natural alleles induce precise changes in the temporal waveform of GI expression, and these changes have detectable effects on phytochrome interacting factor 4 expression and growth. Our findings provide a paradigm for how natural alleles act within day/night cycles to precisely modify temporal gene expression waveforms and cause phenotypic diversity. Such alleles could confer an advantage by adjusting the activity of temporally regulated processes without severely disrupting the circadian system.

Distinct patterns of genetic variation alter flowering responses of Arabidopsis accessions to different daylengths.

Many plants flower in response to seasonal changes in daylength. This response often varies between accessions of a single species. We studied the variation in photoperiod response found in the model species Arabidopsis (Arabidopsis thaliana). Seventy-two accessions were grown under six daylengths varying in 2-h intervals from 6 to 16 h. The typical response was sigmoidal, so that plants flowered early under days longer than 14 h, late under days shorter than 10 h, and at intermediate times under 12-h days. However, many accessions diverged from this pattern and were clustered into groups showing related phenotypes. Thirty-one mutants and transgenic lines were also scored under the same conditions. Statistical comparisons demonstrated that some accessions show stronger responses to different daylengths than are found among the mutants. Genetic analysis of two such accessions demonstrated that different quantitative trait loci conferred an enhanced response to shortening the daylength from 16 to 14 h. Our data illustrate the spectrum of daylength response phenotypes present in accessions of Arabidopsis and demonstrate that similar phenotypic variation in photoperiodic response can be conferred by different combinations of loci.

Effects of genetic perturbation on seasonal life history plasticity.

Like many species, the model plant Arabidopsis thaliana exhibits multiple different life histories in natural environments. We grew mutants impaired in different signaling pathways in field experiments across the species' native European range in order to dissect the mechanisms underlying this variation. Unexpectedly, mutational loss at loci implicated in the cold requirement for flowering had little effect on life history except in late-summer cohorts. A genetically informed photothermal model of progression toward flowering explained most of the observed variation and predicted an abrupt transition from autumn flowering to spring flowering in late-summer germinants. Environmental signals control the timing of this transition, creating a critical window of acute sensitivity to genetic and climatic change that may be common for seasonally regulated life history traits.

Phloem transport of flowering signals.

Seasonal variability in environmental parameters such as day length regulates many aspects of plant development. The transition from vegetative growth to flowering in Arabidopsis is regulated by seasonal changes in day length through a genetically defined molecular cascade known as the photoperiod pathway. Recent advances were made in understanding the tissues in which different components of the photoperiod pathway act to regulate floral induction. These studies highlighted the key role of the FT protein, which is produced in the leaves in response to inductive day lengths and traffics through the phloem to initiate flowering at the shoot apex. Unveiling the cellular and molecular details of this systemic signaling process will be required for a complete understanding of flowering regulation and other photoperiodic processes.

FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis.

In plants, seasonal changes in day length are perceived in leaves, which initiate long-distance signaling that induces flowering at the shoot apex. The identity of the long-distance signal has yet to be determined. In Arabidopsis, activation of FLOWERING LOCUS T (FT) transcription in leaf vascular tissue (phloem) induces flowering. We found that FT messenger RNA is required only transiently in the leaf. In addition, FT fusion proteins expressed specifically in phloem cells move to the apex and move long distances between grafted plants. Finally, we provide evidence that FT does not activate an intermediate messenger in leaves. We conclude that FT protein acts as a long-distance signal that induces Arabidopsis flowering.