Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles for genetically engineered human hepatoma HepG2/8F_HS cells with or without heat treatment, cultured as monolayer or spheroids

ABSTRACT: We developed genetically engineered HepG2/8F_HS cells, in which eight liver-enriched transcription factor (LETF) genes—hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β, HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ— under the control of TRE/PCMVmin promoter were introduced into a previously developed human hepatoma cell line (HepG2-HSP). The heat-inducible synthetic promoter system was introduced into HepG2 cells and tetracycline-responsive transactivator (tTA) and enhanced green fluorescent protein (EGFP) were expressed via positive feedback of tTA transcription in response to heat treatment. HepG2/8F_HS cells can induce high liver functions by heat treatment via overexpression of LETF genes.

ORGANISM(S): Homo sapiens

SUBMITTER: Masamichi Kamihira

PROVIDER: E-MTAB-11486 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MicroRNAs (miRNAs) are an evolutionarily conserved large class of small non-coding RNAs that mediate post-transcriptional silencing of genes and influence a broad spectrum of biological processes ranging from embryonic development to organismal death. Our previous study identified the miR-29 family, three paralogous species of miR-29a/b/c, as the most predominantly expressed small RNA in aged mouse brain compared to neonate one. The mouse brain miR-29 is highly astrocytic. Its expression is quiescent during early brain development, and thereafter steadily increases to a saturating level around the end of the reproductive phase. To explore the functional relevance of miR-29 expression to the neural physiology of mouse brain from the mechanistic perspective of mammalian species-specific lifespan, we here designed a gain-of-function approach through forced expression of miR-29 in astrocyte from mouse fetus and surveyed the resulting alteration in the transcriptional and translational levels. DNA microarray analyses retrieved a total of 5,589 genes showing a temporal significant expression changes in the miR-29-transfected fetal astrocytes, and classified them into two gene groups positively or negatively regulated by miR-29. Mass spectrometry (MS)-based quantification of translational products of miR-29-responsive genes identified 18 species of miR-29 target candidates. We performed functional enrichment analyses using bioinformatics resources to elucidate the biological roles of the gene sets thus identified, and obtained their expression trend that favor the processes for facilitating cell differentiation while supporting normal cell proliferation /survival, which was different from the functional signatures of miR-29 at adult stages, implying the bifunctional property of miR-29 depending on the developmental context. Our present results strongly suggest that miR-29 serves as the central coordinator to shift the global gene expression in the developing mouse brain toward matured phenotype, and through which ensure the programmed transition to the post-developmental stage that is inherently set for the mouse lifespan. DNA microarray experiment: Sample labeling, hybridization and washing were performed following the standard protocol detailed in the Agilent One-Color Microarray-Based Gene Expression Analysis ver.5.7. Briefly, a One-color Spike-Mix was diluted 1000-fold and a 5-μl aliquot of the diluted mix were added to every 0.5 μg of total RNA samples (three each of extraction replicates of miR-29-treated sample and control sample named elsewhere) prior to labeling reaction. The labeling reaction was carried out separately for the RNA samples using a Quick Amp Labeling Kit one-color, in the presence of cyanine 3-CTP. The dye-labeled target (1.6 μg as cRNA) was fragmented and hybridized on an Agilent Whole Mouse Genome 4X44K microarray at 65°C for 17 hr with a Gene Expression Hybridization Kit. The hybridized slide was washed in Gene Expression Wash Buffer 1 at room temperature for 1 min, which was followed by a wash for 1 min in Gene Expression Wash Buffer 2. The processed microarrays were scanned using an Agilent DNA Microarray Scanner. Data extraction from raw image files was done with Agilent Feature Extraction software ver.10.7.

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Gene expression profiles for genetically engineered human hepatoma HepG2/8F_HS cells with or without heat treatment, cultured as monolayer or spheroids

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