Contribution of Intronic miR-338-3p and Its Hosting Gene AATK to Compensatory β-Cell Mass Expansion.

The elucidation of the mechanisms directing β-cell mass regeneration and maintenance is of interest, because the deficit of β-cell mass contributes to diabetes onset and progression. We previously found that the level of the microRNA (miRNA) miR-338-3p is decreased in pancreatic islets from rodent models displaying insulin resistance and compensatory β-cell mass expansion, including pregnant rats, diet-induced obese mice, and db/db mice. Transfection of rat islet cells with oligonucleotides that specifically block miR-338-3p activity increased the fraction of proliferating β-cells in vitro and promoted survival under proapoptotic conditions without affecting the capacity of β-cells to release insulin in response to glucose. Here, we evaluated the role of miR-338-3p in vivo by injecting mice with an adeno-associated viral vector permitting specific sequestration of this miRNA in β-cells. We found that the adeno-associated viral construct increased the fraction of proliferating β-cells confirming the data obtained in vitro. miR-338-3p is generated from an intron of the gene coding for apoptosis-associated tyrosine kinase (AATK). Similarly to miR-338-3p, we found that AATK is down-regulated in rat and human islets and INS832/13 β-cells in the presence of the cAMP-raising agents exendin-4, estradiol, and a G-protein-coupled Receptor 30 agonist. Moreover, AATK expression is reduced in islets of insulin resistant animal models and selective silencing of AATK in INS832/13 cells by RNA interference promoted β-cell proliferation. The results point to a coordinated reduction of miR-338-3p and AATK under insulin resistance conditions and provide evidence for a cooperative action of the miRNA and its hosting gene in compensatory β-cell mass expansion.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Le coeur des ARN non codants - Un long chemin à découvrir [In the heart of noncoding RNA: a long way to go].

The identification and characterization of long noncoding RNA in a variety of tissues represent major achievements that contribute to our understanding of the molecular mechanisms controlling gene expression. In particular, long noncoding RNA play crucial roles in the epigenetic regulation of the adaptive response to environmental cues via their capacity to target chromatin modifiers to specific locus. In addition, these transcripts have been implicated in controlling splicing, translation and degradation of messenger RNA. Long noncoding RNA have also been shown to act as decoy molecules for microRNA. In the heart, a few long noncoding RNA have been demonstrated to regulate cardiac commitment and differentiation during development. Furthermore, recent findings suggest their involvement as regulators of the pathophysiological response to injury in the adult heart. Their high cellular specificity makes them attractive target molecules for innovative therapies and ideal biomarkers.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

Assessing Recent Selection and Functionality at Long Noncoding RNA Loci in the Mouse Genome.

Long noncoding RNAs (lncRNAs) are one of the most intensively studied groups of noncoding elements. Debate continues over what proportion of lncRNAs are functional or merely represent transcriptional noise. Although characterization of individual lncRNAs has identified approximately 200 functional loci across the Eukarya, general surveys have found only modest or no evidence of long-term evolutionary conservation. Although this lack of conservation suggests that most lncRNAs are nonfunctional, the possibility remains that some represent recent evolutionary innovations. We examine recent selection pressures acting on lncRNAs in mouse populations. We compare patterns of within-species nucleotide variation at approximately 10,000 lncRNA loci in a cohort of the wild house mouse, Mus musculus castaneus, with between-species nucleotide divergence from the rat (Rattus norvegicus). Loci under selective constraint are expected to show reduced nucleotide diversity and divergence. We find limited evidence of sequence conservation compared with putatively neutrally evolving ancestral repeats (ARs). Comparisons of sequence diversity and divergence between ARs, protein-coding (PC) exons and lncRNAs, and the associated flanking regions, show weak, but significantly lower levels of sequence diversity and divergence at lncRNAs compared with ARs. lncRNAs conserved deep in the vertebrate phylogeny show lower within-species sequence diversity than lncRNAs in general. A set of 74 functionally characterized lncRNAs show levels of diversity and divergence comparable to PC exons, suggesting that these lncRNAs are under substantial selective constraints. Our results suggest that, in mouse populations, most lncRNA loci evolve at rates similar to ARs, whereas older lncRNAs tend to show signals of selection similar to PC genes.

Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration

Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans.

UNLABELLED: In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. IMPORTANCE: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.

Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana.

In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country.

Tgfbi/Bigh3 silencing activates ERK in mouse retina.

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

Presence of Leishmania RNA Virus 1 in Leishmania guyanensis Increases the Risk of First-Line Treatment Failure and Symptomatic Relapse.

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

Identification of molecular subtypes and gene expression patterns of breast cancer analysing RNA-seq data

Breast cancer is the most common diagnosed cancer and the leading cause of cancer death among females worldwide. It is considered a highly heterogeneous disease and it must be classified into more homogeneous groups. Hence, the purpose of this study was to classify breast tumors based on variations in gene expression patterns derived from RNA sequencing by using different class discovery methods. 42 breast tumors paired-samples were sequenced by Illumine Genome Analyzer and the data was analyzed and prepared by TopHat2 and htseq-count. As reported previously, breast cancer could be grouped into five main groups known as basal epithelial-like group, HER2 group, normal breast-like group and two Luminal groups with a distinctive expression profile. Classifying breast tumor samples by using PAM50 method, the most common subtype was Luminal B and was significantly associated with ESR1 and ERBB2 high expression. Luminal A subtype had ESR1 and SLC39A6 significant high expression, whereas HER2 subtype had a high expression of ERBB2 and CNNE1 genes and low luminal epithelial gene expression. Basal-like and normal-like subtypes were associated with low expression of ESR1, PgR and HER2, and had significant high expression of cytokeratins 5 and 17. Our results were similar compared with TGCA breast cancer data results and with known studies related with breast cancer classification. Classifying breast tumors could add significant prognostic and predictive information to standard parameters, and moreover, identify marker genes for each subtype to find a better therapy for patients with breast cancer.

Interferència mediada per RNA, una nova revolució genètica

Els RNA (o ARN, àcids ribonucleics) són biomolècules lineals de cadena senzilla, com un fil, formades per la unió seqüencial d'altres molècules més senzilles, els nucleòtids. Abans de la descoberta del fenòmen de RNAi es creia que el RNA era només un intermediari silenciós de la maquinària genètica, que transportava cegament les instruccions dels gens, en descodificava el missatge i el convertia en proteïnes, procés que es coneix amb el nom de flux d'informació genètica (del gen, que emmagatzema la informació i és format per ADN, a les proteïnes, que fan la feina especificada pel gen) [...].