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RNA-seq of NPL knock-down in THP1 cells

ABSTRACT: The NPL encodes an enzyme that regulates cellular concentrations of sialic acid (N-acetyl-neuraminic acid) by mediating the reversible conversion of sialic acid into N-acetylmannosamine and pyruvate. As functions of NPL gene in obesity and gluco-metabolic phenotypes is not studied, we knocked down NPL in THP1 cells to understand its roles in modulating the human monocyte-macrophage expression network. Transduction of THP1 cells by NPL-specific lentiviral shRNA stably knocked down its expression at baseline monocytes and in the PMA-induced macrophage state. Global transcriptomic analysis by RNA-seq validated the downregulation of NPL, and comparison of NPL knockdown cells with control-shRNA treated cells further identified 1,183 differentially expressed genes (DEGs). Genes downregulated by the NPL knockdown were significantly enriched for cytokine production, while upregulated genes were enriched for extracellular structure organization. We further compared the effect of macrophage conditioned media (MCM) derived from NPL-shRNA and control-shRNA-expressing THP1 macrophages on SGBS adipocytes. Compared to unconditioned media, MCM derived from either of the THP1 cells differentially regulated key genes involved in adipocyte function and IR. The expression of ADIPOQ, peroxisome proliferator activated receptor gamma (PPARG), and glucose transporter-4 (SLC2A4/GLUT4) was downregulated, while expression of LEP was upregulated in SGBS cells treated with MCM starting from day 4 of the in vitro differentiation. However, PPARG was less repressed and LEP was less activated when SGBS adipocytes were treated on day 4 differentiation with MCM from NPL-shRNA-THP1 cells, suggesting knockdown of NPL partially ameliorated macrophage-induced inflammation of adipocytes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE196888 | GEO | 2022/02/21

REPOSITORIES: GEO

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Project description:The winged helix protein FOXA2 and the nuclear receptor PPARg are highly conserved, regionally-expressed transcription factors that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for FOXA2 in mouse liver and PPARg in mouse adipocytes has previously produced consensus binding sites that are nearly identical to those used by the factors in human cells. Despite this conservation of the canonical binding motif, we report here that the great majority of specific binding regions for FOXA2 in human liver and for PPARg in human adipocytes are not in the orthologous locations to the mouse genome. Nevertheless, gene-centric analysis reveals strong shared transcription factor occupancy near genes in tissue-specific metabolic pathways that are functionally conserved across species. Genes with only species-specific binding sites fail to show enrichment for these pathways. Thus, the biological functions of transcription factors that control specific metabolic functions are highly shared across species. Two TFs, FOXA2 and PPARg, were studied for genome-wide conservation of binding between mouse and human in specific tissues/cell-types (liver for FOXA2, adipocytes for PPARg). The number of replicates for each TF was chosen to obtain a comparable number of reads between the TFs and species. Human FOXA2 ChIP-seq was performed on two biological replicates of human liver samples, in three technical replicates each. Input DNA was also collected and sequenced from both biological samples. Mouse FOXA2 ChIP-seq was performed on four biological replicates of mouse liver samples. The ChIP and sequencing were repeated on two of these biological replicates to create technical replicates for additional sequence reads. Input DNA was sequenced from three additional mouse livers. Human PPARg ChIP-seq was performed on a human adipocyte cell-line (SGBS) differentiated in two replicate cultures. Input DNA was also collected and sequenced from one culture. Mouse PPARg ChIP-seq was performed on 3T3-L1 cells differentiated into adipocytes in culture in a single replicate, and this sequence data was pooled with existing data previously generated by the same lab, already available in GEO (GSE21314). A standard pool of input DNA sample sequence from multiple mouse tissue was used for analyzing the Mouse PPARg ChIP-seq data.

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RNA-seq of NPL knock-down in THP1 cells

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