Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human cell line HepG2 edited with CRISPR and treated with splice-switching oligo inhibiting a pseudoexon in PCCA against control SSO and control HepG2 cells

ABSTRACT: Cellular models of propionic acidemia were generated using HepG2 cells that were edited by CRISPR gene editing to introduce the pathogenic PCCA c.1285-1416A>G variant associated with propionic acidemia, and a 7 bp deletion of a predicted hnRNP A1 binding motif (c.1285-1411delTAGAACA), which causes complete and partial activation of an 84 bp pseudoexon from intron 14 in the PCCA gene, respectively. Treatment by transfection of splice-switching antisense oligonucleotides (SSO) to block inclusion of the PCCA pseudoexon in mRNA, was used to investigate this as a potential therapeutic strategy.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Thomas Doktor

PROVIDER: E-MTAB-13549 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The proinflammatory cytokine, TNFalpha is critical in maintaining liver homeostasis since it is a major determiner of hepatocyte life and death. Considering this, gene transcription profiling was examined in control and TNFalpha treated HepG2 cells. Results indicated that TNFalpha could significantly alter the expression of a significant number of genes; most of them were functionally distributed among molecular functions like catalytic activity, binding, molecular transducer activity, transporter activity, translation and transcription regulator activities or enzyme regulator activity. Also, within genes up-regulated by TNFalpha, several GO terms related to lipid and fat metabolism were significantly overrepresented indicating global dysregulation of fat metabolism within the hepatocyte and those within the down-regulated dataset included genes involved in immunoglobulin receptor activity and IgE binding thereby indicating a compromise in immune defense mechanism(s) apart from those involved the DNA binding and protein binding categories. The interacting network of “lipid metabolism, small molecule biochemistry” was derived to be significantly affected that correlated well with the top canonical pathway of “biosynthesis of steroids” and molecular and cellular function of “lipid metabolism”. All these indicate TNFalpha to be significantly altering the transcriptome profiling within HepG2 cells with genes involved in lipid and steroid metabolism being the most favoured. This study suitably addresses the genes that determine TNFalpha mediated alterations within the hepatocyte mainly the phenotypes of hepatic steatosis and fatty liver that are associated with several hepatic pathological states. HepG2 cells were maintained in DMEM supplemented with 10% fetal calf serum with 1% antibiotic-antimycotic. On attaining confluency, cells were serum starved overnight and incubated in the absence (control) and presence of TNFalpha (0.5nM, 12h). On termination of incubation, total RNA was isolated, reverse transcribed to cDNA and subjected to in vitro transcription to produce biotinylated cRNA that was hybridized to the human array chip (Human Genome U133 Plus 2.0, Affymetrix) according to the manufacturer's instructions. Images were scanned using the GeneChip 3000 7G scanner (Affymetrix) and analysed. The experiment was performed with triplicates of each set (Control and TNF alpha treated). This experiment was reloaded in November 2010 after additional curation.

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RNA-seq of human cell line HepG2 edited with CRISPR and treated with splice-switching oligo inhibiting a pseudoexon in PCCA against control SSO and control HepG2 cells

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