scholarly journals ETMM-07. HYPOXIC REGULATION OF METABOLIC AND STRUCTURAL GENES IN T98 GLIOBLASTOMA MULTIFORME CELLS BY RNA SEQUENCING

2021 ◽  
Vol 3 (Supplement_1) ◽  
pp. i15-i15
Author(s):  
Brian E White ◽  
Edward Liu ◽  
Hakon Hakonarson ◽  
Russell J Buono
Keyword(s):  
Gene Expression ◽  
Glioblastoma Multiforme ◽  
Rna Sequencing ◽  
Therapeutic Potential ◽  
Global Gene Expression ◽  
Total Rna ◽  
Kegg Pathways ◽  
Unfolded Protein ◽  
Dna Replication And Repair ◽  
Significant Enrichment

Abstract Glioblastoma multiforme (GBM) is the most common primary brain cancer and carries a very poor prognosis. The GBM tumor microenvironment is characterized by regions of profound hypoxia, which are associated with a variety of alterations in gene expression that confer survival, proliferation, and resistance to therapy. Multiple mechanisms have been implicated in hypoxia-associated GBM behavior including upregulation of pathways involved in angiogenesis, immunosuppression, and glucose metabolism. Our study aimed to identify changes in gene expression induced by hypoxia among T98G cells via total RNA sequencing. Human T98 GBM cell lines were cultured in a humidified incubator at 37° C and 5% CO2 and were grown in normoxia (21% O2) or hypoxia (95% N2, 5% C02) for 72 hours. Total RNA was harvested, and global gene expression was evaluated via total RNA sequencing. Standard bioinformatics analysis was performed to identify changes in expression associated with hypoxia. Hypoxia in T98 cells led to significant upregulation of genes implicated in canonical glycolysis, focal adhesion, extracellular matrix reorganization, and endoplasmic reticulum-associated protein processing. We document 690 genes and 11 associated KEGG pathways that demonstrated significant enrichment (p ≤0.01 with Bonferroni, Benjamini, and False Discovery Rate corrections) induced by hypoxia. Notably, upregulation of the IRE1-mediated unfolded protein response was observed. DrugBank database analysis identified four molecules targeting genes upregulated in hypoxic T98G cells: tenecteplase (p = 0.013, 5 gene targets), succinic acid (p = 0.02, 7 targets), artenimol (p = 0.013, 13 targets), and copper (p = 0.0015, 22 targets). We document 733 genes and 6 associated KEGG pathways significantly downregulated (p ≤0.01) in hypoxia, including genes associated with DNA replication and repair, mitotic processes, and spliceosome function. Total RNA sequencing showed hypoxic upregulation of genes involved in various pathways associated with neoplastic GBM behavior and identified multiple candidate molecules which may hold therapeutic potential.

scholarly journals Effect of Bortezomib on Global Gene Expression in PC12-Derived Nerve Cells

2020 ◽  
Vol 21 (3) ◽  
pp. 751 ◽  
Author(s):  
Karolina Łuczkowska ◽  
Dorota Rogińska ◽  
Zofia Ulańczyk ◽  
Bogusław Machaliński
Keyword(s):  
Gene Expression ◽  
Mirna Expression ◽  
Global Gene Expression ◽  
Nerve Cells ◽  
Microarray Expression Data ◽  
Neurotransmitter Secretion ◽  
Dna Replication And Repair ◽  
Gene Correlation ◽  
Regulation Of Cell Cycle ◽  
Microarray Expression

Peripheral neuropathy is one of the main side-effects of novel therapeutics used in oncohematological diseases, but the molecular basis underlying its development and progression as well as neurotoxicity mechanisms induced by the use of these therapeutics are still not fully elucidated. The aim of this study was to demonstrate the effect of bortezomib on global gene and miRNA expression on PC12-derived nerve cells. Microarray analysis showed that expression of 1383 genes was downregulated at least two fold and 671 genes were upregulated at least two fold in PC12-derived nerve cells treated with bortezomib compared to untreated/control cells. Analysis of functional annotations mainly identified downregulated processes (e.g., regulation of cell cycle, DNA replication and repair, regulation of cell migration, neuron projection morphogenesis and neurotransmitter secretion). The result of miRNA expression analysis demonstrated only 11 significantly downregulated miRNAs (at least two fold) in bortezomib-treated PC12-derived nerve cells vs. control cells. MiRNAs regulate gene expression, therefore we decided to conduct an analysis comparing the outcomes of miRNA microarray expression data to the obtained mRNA data. The most interesting miRNA–target gene correlation is downregulated expression of miR-130a-3p and miR-152-3p and as a result of this downregulation the expression of the Gadd45 increased. This gene is a member of a group of genes, the transcript expression of which is enhanced after stressful growth arrest conditions and treatment with DNA-damaging agents like drugs or mutagens.

scholarly journals Global Gene Expression and Mutation Signatures Are Preserved in PDX Models of Pediatric AML and Aid Discovery of Targeted Therapy for Cases with CBFA2T3/GLIS2 Rearrangement

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3766-3766
Author(s):  
Mark Wunderlich ◽  
Jing Chen ◽  
Eric O'Brien ◽  
Nicole Manning ◽  
Christina Sexton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Targeted Therapies ◽  
Global Gene Expression ◽  
Specific Gene ◽  
Sequencing Data ◽  
Patient Sample ◽  
Nsg Mice ◽  
Pediatric Aml ◽  
Pdx Models

Therapies for pediatric acute myeloid leukemia (AML) remain unsatisfactory and generally do not incorporate molecularly-targeted agents aside from FLT3 inhibitors outside of the relapse setting. Patient-derived xenograft (PDX) models of AML are increasingly accessible for the preclinical evaluation of targeted therapies, though the degree to which these systems recapitulate the disease state as found in patients has not been well defined for AML. Gene expression profiling of patient blasts has been successfully used to discriminate distinct subtypes of AML, to uncover sub-type specific vulnerabilities, and to predict response to therapy and outcomes. We sought to systematically examine PDX models of pediatric AML for their ability to replicate global gene expression patterns and preserve mutational signatures found in patients. In addition, we conducted in-depth bioinformatic analyses of samples with cryptic CBA2T3-GLIS2 fusion generated by the inv(16)(p13.3q24.3) for identification of potential novel targeted therapies. We performed detailed analyses of RNA sequencing data from a diverse series of 24 pediatric AML PDX models established from samples obtained from patients with relapse and refractory disease. Initially we compared our PDX data against 49 selected relapse and refractory patient sample data files found in the NCI TARGET dataset of pediatric AML. When applying unsupervised hierarchical clustering to the PDX samples, we found that clustering was associated with MLL status. Clustering of the combined sets of samples by MLL status showed integration of samples according to mutation profile, regardless of data source (PDX or patient). The expression levels of all detectable transcripts were highly conserved between PDX and patient MLL-r samples. Separate analysis of each dataset yielded MLL specific gene lists that included a subset of overlapping genes which may point to a unique relapse and refractory pediatric MLL-r signature. This list contains several interesting new targets for further study. A subset of 12 PDX models were compared directly to the matched patient sample from which they were established. This analysis revealed strong similarity, with each PDX most closely related to its matched patient sample, suggesting retention of sample-specific gene expression in immune deficient mice. We set up our PDX models in NSG mice with transgenic expression of human myelo-supportive cytokines SCF, GM-CSF, and IL-3 in order to promote the most efficient and robust engraftment of precious patient material. In order to detect any skewing effects due to the host mouse strain, we compared NSGS PDX RNA sequencing data to 10 matched NSG PDX models. This comparison revealed consistent differences in only 9 transcripts, which were almost entirely related to increased JAK/STAT signaling and macrophage activation pathways in NSGS mice relative to NSG mice. Interestingly, during this analysis we observed a distinct PCA-driven clustering of a pair of PDX samples with previously clinically unidentified driver mutations. Reanalysis of the RNA sequencing data revealed evidence of a cryptic GLIS2 rearrangement (found in ~1% of pediatric AML cases) as the driver mutation, which was subsequently confirmed by RT-PCR in both samples. The unique CBFA2T3/GLIS2 RNA signature was mined to guide the composition of a focused 75-molecule in vitro drug screen against ex vivo PDX samples with an emphasis on the SHH, WNT, and BCL2 pathways. This screen identified the Wnt-C59 PORCN inhibitor as having specific activity against CBFA2T3/GLIS2+ AMLs. Further testing of C-59 in combinatorial studies revealed enhanced effects with the addition of the BCL2 inhibitor, venetoclax. In vivo experiments are currently underway to determine the pre-clinical efficacy of this novel combination. In summary, we found highly significant fidelity of gene expression in PDX models of relapse and refractory pediatric AML. Analysis of this dataset has led to several insights, including potential targeted therapies, highlighting how this system could be a valuable tool for discovery of novel targeted therapies, especially for very rare, distinct subtypes of disease. Disclosures Perentesis: Kurome Therapeutics: Consultancy.

scholarly journals Transcriptome Changes in the Mink Uterus during Blastocyst Dormancy and Reactivation

2019 ◽  
Vol 20 (9) ◽  
pp. 2099
Author(s):  
Xinyan Cao ◽  
Jiaping Zhao ◽  
Yong Liu ◽  
Hengxing Ba ◽  
Haijun Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Embryo Implantation ◽  
Global Gene Expression ◽  
Gene Clusters ◽  
Protein Protein Interaction ◽  
Kegg Pathways ◽  
Maternal Regulation

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.

scholarly journals Gene Expression Profile of Human Mesenchymal Stromal Cells Exposed to Hypoxic and Pseudohypoxic Preconditioning—An Analysis by RNA Sequencing

2021 ◽  
Vol 22 (15) ◽  
pp. 8160
Author(s):  
Katarzyna Zielniok ◽  
Anna Burdzinska ◽  
Victor Murcia Pienkowski ◽  
Agnieszka Koppolu ◽  
Malgorzata Rydzanicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Therapeutic Potential ◽  
Phospholipid Metabolism ◽  
Functional Enrichment ◽  
Valuable Insight ◽  
Illumina Platform ◽  
Insight Into

Mesenchymal stromal cell (MSC) therapy is making its way into clinical practice, accompanied by research into strategies improving their therapeutic potential. Preconditioning MSCs with hypoxia-inducible factors-α (HIFα) stabilizers is an alternative to hypoxic priming, but there remains insufficient data evaluating its transcriptomic effect. Herein, we determined the gene expression profile of 6 human bone marrow-derived MSCs preconditioned for 6 h in 2% O2 (hypoxia) or with 40 μM Vadadustat, compared to control cells and each other. RNA-Sequencing was performed using the Illumina platform, quality control with FastQC and adapter-trimming with BBDUK2. Transcripts were mapped to the Homo_sapiens. GRCh37 genome and converted to relative expression using Salmon. Differentially expressed genes (DEGs) were generated using DESeq2 while functional enrichment was performed in GSEA and g:Profiler. Comparison of hypoxia versus control resulted in 250 DEGs, Vadadustat versus control 1071, and Vadadustat versus hypoxia 1770. The terms enriched in both phenotypes referred mainly to metabolism, in Vadadustat additionally to vesicular transport, chromatin modifications and interaction with extracellular matrix. Compared with hypoxia, Vadadustat upregulated autophagic, phospholipid metabolism, and TLR cascade genes, downregulated those of cytoskeleton and GG-NER pathway and regulated 74 secretory factor genes. Our results provide valuable insight into the transcriptomic effects of these two methods of MSCs preconditioning.

scholarly journals Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants

2021 ◽  
Author(s):  
S Cole Kitzman ◽  
Tingting Duan ◽  
Miles A Pufall ◽  
Pamela K Geyer
Keyword(s):  
Gene Expression ◽  
Nuclear Lamina ◽  
Global Gene Expression ◽  
Regulation Of Transcription ◽  
Germline Stem Cells ◽  
General Role ◽  
Lamin B ◽  
Expression Of Genes ◽  
Dna Replication And Repair ◽  
Protein Barrier

Abstract The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin binding protein Barrier-to-autointegration factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to deformation of NL structure and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf mis-regulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained mis-expressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global mis-expression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found up-regulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

scholarly journals The Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy

Circulation ◽  
2021 ◽  
Author(s):  
Dries A.M. Feyen ◽  
Isaac Perea-Gil ◽  
Renee G.C. Maas ◽  
Magdalena Harakalova ◽  
Alexandra A. Gavidia ◽  
...  
Keyword(s):  
Single Cell ◽  
Unfolded Protein Response ◽  
Rna Sequencing ◽  
Therapeutic Potential ◽  
Compensatory Mechanism ◽  
Unfolded Protein ◽  
Single Cell Rna Sequencing ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
The Impact

Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy (DCM) with a high prevalence of ventricular arrhythmias. How the R14 deletion causes DCM is poorly understood and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease-mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We utilized both 2D and 3D functional contractility assays to evaluate the impact of modulating disease relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro . Single-cell RNA sequencing revealed the induction of the unfolded protein response pathway (UPR) in PLN R14del compared to isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the three main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP protein Inducer X (BiX). PLN R14del hiPSC-CMs treated with BiX showed a dose-dependent amelioration of the contractility deficit of in both 2D cultures and 3D engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

scholarly journals A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Simon Haile ◽  
Richard D. Corbett ◽  
Veronique G. LeBlanc ◽  
Lisa Wei ◽  
Stephen Pleasance ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput Sequencing ◽  
Single Cells ◽  
Cell Types ◽  
Full Length ◽  
Sequencing Data ◽  
Total Rna ◽  
Specific Protocol

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

scholarly journals Transcriptome RNA Sequencing Data Set of Gene Expression in Moraxella catarrhalis On- and Off-Phase Variants of the Type III DNA Methyltransferase ModM3

2020 ◽  
Vol 9 (14) ◽  
Author(s):  
Luke V. Blakeway ◽  
Aimee Tan ◽  
Ian R. Peak ◽  
John M. Atack ◽  
Kate L. Seib
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Moraxella Catarrhalis ◽  
Dna Methyltransferase ◽  
Global Gene Expression ◽  
Chronic Obstructive ◽  
Sequencing Data ◽  
Data Set ◽  
Obstructive Pulmonary Disease ◽  
Content Type

Moraxella catarrhalis is a leading bacterial cause of otitis media and exacerbations of chronic obstructive pulmonary disease. Here, we announce a transcriptome RNA sequencing data set detailing global gene expression in two M. catarrhalis CCRI-195ME variants with expression of the DNA methyltransferase ModM3 phase varied either on or off.

scholarly journals RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle–mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie’s disease penile tunica albuginea

BMC Urology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Guo Nan Yin ◽  
Shuguang Piao ◽  
Zhiyong Liu ◽  
Lei Wang ◽  
Jiyeon Ock ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Tunica Albuginea ◽  
Global Gene Expression ◽  
Extracellular Vesicle ◽  
Peyronie’S Disease ◽  
Rt Pcr ◽  
Cultured Fibroblasts ◽  
Primary Fibroblasts ◽  
Peyronie's Disease

Abstract Background Peyronie’s disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)–mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV–mimetic NVs (PC–NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC–NVs in primary fibroblasts derived from human PD plaque. Methods Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)–mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC–NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. Results A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC–NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. Conclusion The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

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