The long noncoding RNA MALAT1 promotes tumor-driven angiogenesis by up-regulating pro-angiogenic gene expression

Neuroblastoma is the most common solid tumor during early childhood. One of the key features of neuroblastoma is extensive tumor-driven angiogenesis due to hypoxia. However, the mechanism through which neuroblastoma cells drive angiogenesis is poorly understood. Here we show that the long noncoding RNA MALAT1 was upregulated in human neuroblastoma cell lines under hypoxic conditions. Conditioned media from neuroblastoma cells transfected with small interfering RNAs (siRNA) targeting MALAT1, compared with conditioned media from neuroblastoma cells transfected with control siRNAs, induced significantly less endothelial cell migration, invasion and vasculature formation. Microarray-based differential gene expression analysis showed that one of the genes most significantly downregulated following MALAT1 suppression in human neuroblastoma cells under hypoxic conditions was fibroblast growth factor 2 (FGF2). RT-PCR and immunoblot analyses confirmed that MALAT1 suppression reduced FGF2 expression, and Enzyme-Linked Immunosorbent Assays revealed that transfection with MALAT1 siRNAs reduced FGF2 protein secretion from neuroblastoma cells. Importantly, addition of recombinant FGF2 protein to the cell culture media reversed the effects of MALAT1 siRNA on vasculature formation. Taken together, our data suggest that up-regulation of MALAT1 expression in human neuroblastoma cells under hypoxic conditions increases FGF2 expression and promotes vasculature formation, and therefore plays an important role in tumor-driven angiogenesis

A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy.

Early infantile epileptic encephalopathies (EOEE) are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl-]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a "change-of-function" KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient

Mouse Model of Mutated in Colorectal Cancer Gene Deletion Reveals Novel Pathways in Inflammation and Cancer

© 2019 The Authors Background & Aims: The early events by which inflammation promotes cancer are still not fully defined. The MCC gene is silenced by promoter methylation in colitis-associated and sporadic colon tumors, but its functional significance in precancerous lesions or polyps is not known. Here, we aimed to determine the impact of Mcc deletion on the cellular pathways and carcinogenesis associated with inflammation in the mouse proximal colon. Methods: We generated knockout mice with deletion of Mcc in the colonic/intestinal epithelial cells (MccΔIEC) or in the whole body (MccΔ/Δ). Drug-induced lesions were analyzed by transcriptome profiling (at 10 weeks) and histopathology (at 20 weeks). Cell-cycle phases and DNA damage proteins were analyzed by flow cytometry and Western blot of hydrogen peroxide–treated mouse embryo fibroblasts. Results: Transcriptome profiling of the lesions showed a strong response to colon barrier destruction, such as up-regulation of key inflammation and cancer-associated genes as well as 28 interferon γ–induced guanosine triphosphatase genes, including the homologs of Crohn's disease susceptibility gene IRGM. These features were shared by both Mcc-expressing and Mcc-deficient mice and many of the altered gene expression pathways were similar to the mesenchymal colorectal cancer subtype known as consensus molecular subtype 4 (CMS4). However, Mcc deletion was required for increased carcinogenesis in the lesions, with adenocarcinoma in 59% of MccΔIEC compared with 19% of Mcc-expressing mice (P =.002). This was not accompanied by hyperactivation of β-catenin, but Mcc deletion caused down-regulation of DNA repair genes and a disruption of DNA damage signaling. Conclusions: Loss of Mcc may promote cancer through a failure to repair inflammation-induced DNA damage. We provide a comprehensive transcriptome data set of early colorectal lesions and evidence for the in vivo significance of MCC silencing in colorectal cancer

Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent

The epidermal compartment is complex and organized into several strata composed of keratinocytes (KCs), including basal, spinous, granular, and corniWed layers. The continuous process of self-renewal and barrier formation is dependent on a homeostatic balance achieved amongst KCs involving proliferation, diVerentiation, and cell death. To determine genes responsible for initiating and maintaining a corniWed epidermis, organotypic cultures comprised entirely of stratiWed KCs creating epidermal equivalents (EE) were raised from a submerged state to an air/liquid (A/L) interface. Compared to the array proWle of submerged cultures containing KCs predominantly in a proliferative (relatively undiVerentiated) state, EEs raised to an A/L interface displayed a remarkably consistent and distinct proWle of mRNAs. Cultures lifted to an A/L interface triggered the induction of gene groups that regulate proliferation, diVerentiation, and cell death. Next, diVerentially expressed microRNAs (miRNAs) and long noncoding (lncRNA) RNAs were identiWed in EEs. Several diVerentially expressed miRNAs were validated by qRT-PCR and Northern blots. miRNAs 203, 205 and Let-7b were up-regulated at early time points (6, 18 and 24 h) but downregulated by 120 h. To study the lncRNA regulation in EEs, we proWled lncRNA expression by microarray and validated the results by qRT-PCR. Although the diVerential expression of several lncRNAs is suggestive of a role in epidermal diVerentiation, their biological functions remain to be elucidated. The current studies lay the foundation for relevant model systems to address such fundamentally important biological aspects of epidermal structure and function in normal and diseased human skin

SEQADAPT: an adaptable system for the tracking, storage and analysis of high throughput sequencing experiments

<p>Abstract</p> <p>Background</p> <p>High throughput sequencing has become an increasingly important tool for biological research. However, the existing software systems for managing and processing these data have not provided the flexible infrastructure that research requires.</p> <p>Results</p> <p>Existing software solutions provide static and well-established algorithms in a restrictive package. However as high throughput sequencing is a rapidly evolving field, such static approaches lack the ability to readily adopt the latest advances and techniques which are often required by researchers. We have used a loosely coupled, service-oriented infrastructure to develop SeqAdapt. This system streamlines data management and allows for rapid integration of novel algorithms. Our approach also allows computational biologists to focus on developing and applying new methods instead of writing boilerplate infrastructure code.</p> <p>Conclusion</p> <p>The system is based around the Addama service architecture and is available at our website as a demonstration web application, an installable single download and as a collection of individual customizable services.</p

Integrating exome sequencing into a diagnostic pathway for epileptic encephalopathy: Evidence of clinical utility and cost effectiveness

© 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Background: Epileptic encephalopathies are a devastating group of neurological conditions in which etiological diagnosis can alter management and clinical outcome. Exome sequencing and gene panel testing can improve diagnostic yield but there is no cost-effectiveness analysis of their use or consensus on how to best integrate these tests into clinical diagnostic pathways. Methods: We conducted a retrospective cost-effectiveness study comparing trio exome sequencing with a standard diagnostic approach, for a well-phenotyped cohort of 32 patients with epileptic encephalopathy, who remained undiagnosed after “first-tier” testing. Sensitivity analysis was included with a range of commercial exome and multigene panels. Results: The diagnostic yield was higher for the exome sequencing (16/32; 50%) than the standard arm (2/32; 6.2%). The trio exome sequencing pathway was cost-effective compared to the standard diagnostic pathway with a cost saving of AU$5,236 (95$2,482; $9,784) per additional diagnosis; the standard pathway cost approximately 10 times more per diagnosis. Sensitivity analysis demonstrated that the majority of commercial exome sequencing and multigene panels studied were also cost-effective. The clinical utility of all diagnoses was reported. Conclusion: Our study supports the integration of exome sequencing and gene panel testing into the diagnostic pathway for epileptic encephalopathy, both in terms of cost effectiveness and clinical utility. We propose a diagnostic pathway that integrates initial rapid screening for treatable causes and comprehensive genomic screening. This study has important implications for health policy and public funding for epileptic encephalopathy and other neurological conditions

Examples of sequence conservation analyses capture a subset of mouse long non-coding RNAs sharing homology with fish conserved genomic elements

Background: Long non-coding RNAs (lncRNA) are a major class of non-coding RNAs. They are involved in diverse intra-cellular mechanisms like molecular scaffolding, splicing and DNA methylation. Through these mechanisms they are reported to play a role in cellular differentiation and development. They show an enriched expression in the brain where they are implicated in maintaining cellular identity, homeostasis, stress responses and plasticity. Low sequence conservation and lack of functional annotations make it difficult to identify homologs of mammalian lncRNAs in other vertebrates. A computational evaluation of the lncRNAs through systematic conservation analyses of both sequences as well as their genomic architecture is required.Results: Our results show that a subset of mouse candidate lncRNAs could be distinguished from random sequences based on their alignment with zebrafish phastCons elements. Using ROC analyses we were able to define a measure to select significantly conserved lncRNAs. Indeed, starting from ~2,800 mouse lncRNAs we could predict that between 4 and 11% present conserved sequence fragments in fish genomes. Gene ontology (GO) enrichment analyses of protein coding genes, proximal to the region of conservation, in both organisms highlighted similar GO classes like regulation of transcription and central nervous system development. The proximal coding genes in both the species show enrichment of their expression in brain. In summary, we show that interesting genomic regions in zebrafish could be marked based on their sequence homology to a mouse lncRNA, overlap with ESTs and proximity to genes involved in nervous system development.Conclusions: Conservation at the sequence level can identify a subset of putative lncRNA orthologs. The similar protein-coding neighborhood and transcriptional information about the conserved candidates provide support to the hypothesis that they share functional homology. The pipeline herein presented represents a proof of principle showing that a portion between 4 and 11% of lncRNAs retains region of conservation between mammals and fishes. We believe this study will result useful as a reference to analyze the conservation of lncRNAs in newly sequenced genomes and transcriptomes. \uc2\ua9 2013 Basu et al.; licensee BioMed Central Ltd

A comparative genome-wide study of ncRNAs in trypanosomatids

<p>Abstract</p> <p>Background</p> <p>Recent studies have provided extensive evidence for multitudes of non-coding RNA (ncRNA) transcripts in a wide range of eukaryotic genomes. ncRNAs are emerging as key players in multiple layers of cellular regulation. With the availability of many whole genome sequences, comparative analysis has become a powerful tool to identify ncRNA molecules. In this study, we performed a systematic genome-wide in silico screen to search for novel small ncRNAs in the genome of <it>Trypanosoma brucei </it>using techniques of comparative genomics.</p> <p>Results</p> <p>In this study, we identified by comparative genomics, and validated by experimental analysis several novel ncRNAs that are conserved across multiple trypanosomatid genomes. When tested on known ncRNAs, our procedure was capable of finding almost half of the known repertoire through homology over six genomes, and about two-thirds of the known sequences were found in at least four genomes. After filtering, 72 conserved unannotated sequences in at least four genomes were found, 29 of which, ranging in size from 30 to 392 nts, were conserved in all six genomes. Fifty of the 72 candidates in the final set were chosen for experimental validation. Eighteen of the 50 (36%) were shown to be expressed, and for 11 of them a distinct expression product was detected, suggesting that they are short ncRNAs. Using functional experimental assays, five of the candidates were shown to be novel H/ACA and C/D snoRNAs; these included three sequences that appear as singletons in the genome, unlike previously identified snoRNA molecules that are found in clusters. The other candidates appear to be novel ncRNA molecules, and their function is, as yet, unknown.</p> <p>Conclusions</p> <p>Using comparative genomic techniques, we predicted 72 sequences as ncRNA candidates in <it>T. brucei</it>. The expression of 50 candidates was tested in laboratory experiments. This resulted in the discovery of 11 novel short ncRNAs in procyclic stage <it>T. brucei</it>, which have homologues in the other trypansomatids. A few of these molecules are snoRNAs, but most of them are novel ncRNA molecules. Based on this study, our analysis suggests that the total number of ncRNAs in trypanosomatids is in the range of several hundred.</p