High-throughput identification of protein functional similarities using a gene-expression-based siRNA screen

Abstract Background Human T-lymphotropic virus 1 (HTLV-1) infection may lead to the development of Adult T-cell leukemia/lymphoma (ATLL). To further elucidate the pathophysiology of this aggressive CD4+ T-cell malignancy, we have performed an integrated systems biology approach to analyze previous transcriptome datasets focusing on differentially expressed miRNAs (DEMs) in peripheral blood of ATLL patients. Methods Datasets GSE28626, GSE31629, GSE11577 were used to identify ATLL-specific DEM signatures. The target genes of each identified miRNA were obtained to construct a protein-protein interactions network using STRING database. The target gene hubs were subjected to further analysis to demonstrate significantly enriched gene ontology terms and signaling pathways. Quantitative reverse transcription Polymerase Chain Reaction (RTqPCR) was performed on major genes in certain pathways identified by network analysis to highlight gene expression alterations. Results High-throughput in silico analysis revealed 9 DEMs hsa-let-7a, hsa-let-7g, hsa-mir-181b, hsa-mir-26b, hsa-mir-30c, hsa-mir-186, hsa-mir-10a, hsa-mir-30b, and hsa-let-7f between ATLL patients and healthy donors. Further analysis revealed the first 5 of DEMs were directly associated with previously identified pathways in the pathogenesis of HTLV-1. Network analysis demonstrated the involvement of target gene hubs in several signaling cascades, mainly in the MAPK pathway. RT-qPCR on human ATLL samples showed significant upregulation of EVI1, MKP1, PTPRR, and JNK gene vs healthy donors in MAPK/JNK pathway. Discussion The results highlighted the functional impact of a subset dysregulated microRNAs in ATLL on cellular gene expression and signal transduction pathways. Further studies are needed to identify novel biomarkers to obtain a comprehensive mapping of deregulated biological pathways in ATLL.

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Fast and Efficient 5′P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis

Plants ◽

10.3390/plants10030466 ◽

2021 ◽

Vol 10 (3) ◽

pp. 466

Author(s):

Marie-Christine Carpentier ◽

Cécile Bousquet-Antonelli ◽

Rémy Merret

Keyword(s):

Gene Expression ◽

Quality Control ◽

Transcriptional Regulation ◽

High Throughput ◽

Library Preparation ◽

Working Day ◽

Set Up ◽

Post Transcriptional Regulation ◽

Commercial Kit

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5′monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5′P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5′P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

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Co-expressional conservation in virulence and stress related genes of three Gammaproteobacterial species: Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa

Molecular BioSystems ◽

10.1039/c5mb00353a ◽

2015 ◽

Vol 11 (11) ◽

pp. 3137-3148

Author(s):

Nazanin Hosseinkhan ◽

Peyman Zarrineh ◽

Hassan Rokni-Zadeh ◽

Mohammad Reza Ashouri ◽

Ali Masoudi-Nejad

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Systems Biology ◽

Gene Expression Data ◽

High Throughput ◽

Expression Data ◽

P Gene ◽

Stress Related Genes ◽

High Throughput Gene Expression

Gene co-expression analysis is one of the main aspects of systems biology that uses high-throughput gene expression data.

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High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors

International Journal of Molecular Sciences ◽

10.3390/ijms22063022 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3022

Author(s):

Tatjana Ullmann ◽

Sonja Luckhardt ◽

Markus Wolf ◽

Michael J. Parnham ◽

Eduard Resch

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

High Throughput ◽

Transcription Initiation ◽

High Throughput Screening ◽

Inflammatory Responses ◽

Hdac Inhibitors ◽

Screening Assay ◽

Bet Inhibitors ◽

Anti Inflammatory

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

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Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a46 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Kasey C Vickers ◽

Michael G Levin ◽

Michael P Anderson ◽

Qing Xu ◽

Joshua Anzinger ◽

...

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

High Throughput ◽

Small Rna ◽

Cellular Response ◽

Culture Media ◽

Recipient Cell ◽

Inflammatory Cells ◽

High Density Lipoproteins ◽

Small Rna Sequencing

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.

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Abstract 12905: Gene Expression and Expression Quantifying Trait Loci Analysis of Left Atrium of Patients With Post-operative Atrial Fibrillation

Circulation ◽

10.1161/circ.132.suppl_3.12905 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Martin I Sigurdsson ◽

Mahyar Heydarpour ◽

Louis Saddic ◽

Tzuu-Wang Chang ◽

Stanton K Shernan ◽

...

Keyword(s):

Gene Expression ◽

Atrial Fibrillation ◽

Left Atrium ◽

High Throughput ◽

Specific Gene ◽

Eqtl Analysis ◽

Gene Variants ◽

Nucleotide Polymorphisms ◽

Novel Genes ◽

Trait Loci

Introduction: The majority of information on the genetic background of atrial fibrillation (AF) results from genomic DNA variant analysis without consideration of tissue expression. Hypothesis: Analysis of tissue-specific gene expression in left atrium (LA) can further understanding of the molecular mechanism of identified AF risk variants, and identify novel genes and gene variants associated with AF. Methods: We isolated mRNA from samples of the LA free wall taken during mitral valve surgery in 62 Caucasian individuals. Gene expression in the LA was compared between patients who did and did not have post-operative AF (poAF) using high-throughput RNA expression. Using genotypes of 1.4 million single nucleotide polymorphisms (SNP) we performed cis expression quantifying trait loci (eQTL) analysis, correlating gene expression of each gene with the genotypes of adjacent (<1Mbp) SNPs. Results: We identified 23 differentially expressed genes in the LA of patients with poAF, including three potassium channel genes (KCNA7, KCNH8 and KCNK17). The largest expression difference was in LOC645323, a long non-coding RNA. The expression of PITX2, ZFHX3 and KCNN3, previously shown to be associated with AF, did not differ between patients with and without poAF. We identified 12,476 cis eQTL relationships in the LA, several of those included genetic regions and genes previously associated with AF. We confirmed an eQTL relationship between rs3744029 genotype and the expression of MYOZ1. Furthermore we describe a novel eQTL relationship between rs6795970 genotype and the expression of the SCN10A gene. Conclusions: We have analysed the human LA expression via high-throughput RNA sequencing, and identified novel genes and gene variants likely involved in the molecular pathophysiology of AF.

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