Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Investigation of Gene Expression Changes in Human Umbilical Vein Endothelial cells (HUVECs) following SLC25A13 Knockdown via shRNA Lentiviral Transduction

ABSTRACT: In this study, we discover an essential role for the mitochondrial glutamate-aspartate antiporter SLC25A13 in biomass synthesis in endothelial cells (ECs). To delineate the global changes of SLC25A13 silencing at a molecular level, we performed high-throughput bulk RNA-sequencing and analyzed the differences between the gene expression profiles of CTRL and SLC25A13-KD ECs. HUVECs were transduced at a multiplicity of infection (MOI) of 30. Transductions were performed on day 0 and after 24 hours the cells were refed with fresh medium. RNA of scrambled control and SLC25A13-KD ECs were extracted 6 days after seeding. Samples were indexed to allow for multiplexing. Libraries were sequenced on an Illumina NovaSeq6000 platform. Single-end reads of 100 bp length were produced with a minimum of 20 M reads per sample. The reads were aligned using a standard pipeline for splice-aware alignment. The reads per gene were then quantified. The study accounted for 12 samples (6 for CTRL and 6 for SLC25A13-KD ECs).

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Abhishek Subramanian

PROVIDER: E-MTAB-14591 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure and increased susceptibility to cancer. Of the fifteen FA proteins, Fanconi anemia group C (FANCC) is one of eight FA core complex components of the FA pathway. Unlike other FA core complex proteins, FANCC is mainly localized in the cytoplasm, where it is thought to function in apoptosis, redox regulation, cytokine signaling and other processes. Previously, we showed that regulation of FANCC involved proteolytic processing during apoptosis. To elucidate the biological significance of this proteolytic modification, we searched for molecular interacting partners of proteolytic FANCC fragments. Among the candidates obtained, the transcriptional corepressor protein C-terminal binding protein-1 (CtBP1) interacted directly with FANCC and other FA core complex proteins. Although not required for stability of the FA core complex or ubiquitin ligase activity, CtBP1 is essential for proliferation, cell survival and maintenance of chromosomal integrity. Expression profiling of CtBP1-depleted and FA-depleted cells revealed that several genes were commonly up- and down-regulated, including the Wnt antagonist Dickkopf-1 (DKK1). These findings suggest that FA and Wnt signaling via CtBP1 could share common effectors. HeLa cell line was grown in DMEM media supplemented with 10% FBS and were incubated in 5% CO2 at 37Â°C. A four-plasmid (pRSV-Rev, pMDLg/pRRE, pMD2.G and pLKO.1) expression system was used for lentiviral production. Different pLKO.1 plasmids carrying shRNAs targeting FANCA, FANCD2, CtBP1 or CtBP2 or a lentiviral control vector pLKO.1-scrambled were used. Lentiviral particles were produced via calcium phosphate-mediated transient transfection of the four plasmids into HEK293T cells. Cells were exposed to appropriate lentiviral particles. In each experiment, HeLa cells were transduced for 6 hours with filtered supernatant containing recombinant lentiviral particles. After transduction, the cells were cultured for 72 hours. Total RNA extracts from 3 different samples of each scrambled, CtBPs and FANCD2 shRNA treated HeLa cells were subjected to gene expression profiling via microarray analysis. Gene expression profiles were determined with Affymetrix GeneChipÂ® Human Gene 1.0.

Project description:Background: ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation, whereas its role in leukemia propagation and maintenance remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines. Findings: Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-DOWN set was significantly enriched for genes included in the cell activation, immune response, apoptosis, signal transduction and development and differentiation categories, whereas in the E/R KD-UP set only the PI3K/AKT/mTOR signaling and hematopoietic stem cells categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories stem cell properties, B-cell differentiation, immune response, cell adhesion and DNA damage with RT-qPCR. Conclusion: The results of our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with regular B-cell development by repressing key functions that are necessary under physiological circumstances. E/R may thus constitute also the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets. Knockdown of ETV6/RUNX1 in two t(12;21) positive leukemic cell lines

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Investigation of Gene Expression Changes in Human Umbilical Vein Endothelial cells (HUVECs) following SLC25A13 Knockdown via shRNA Lentiviral Transduction

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