ShRNA profiling of human hepatocyte HepG2 cells knocked down for HNF4 to investigate its role in inflammatory response
ABSTRACT: To obtain a genomic view of hepatocyte nuclear factor-4α (HNF-4α) in the regulation of the inflammatory response, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokines in a model of knock-down HNF-4α HepG2 cells. The results indicate an extensive role for HNF-4α plays in the regulation of a large number of the liver-specific genes. Majority of genes (71%) affected by cytokine treatment are also affected by HNF-4α knock-down. This significant overlap suggests that HNF-4α may play a role in regulating the cytokine-induced inflammatory response. Experiment Overall Design: The different treated HepG2 cells were grouped into 4 groups (four replicates in each group): Group1, Control; Group2, HNF-4α shRNA treated cells; Group3, cytokine treated group; Group4, HNF-4α shRNA and cytokine treatments. RNA extraction and hybridization on Affymetrix microarrays were processed. The expression profiles between different groups were analyzed and compared. In order to explore the function of HNF-4α in the inflammatory response, a set of 170 genes annotated as inflammatory response was obtained from GO (geneotology.org), the enrichments of these inflammatory genes were analyzed in different treated groups.
Project description:To obtain a genomic view of hepatocyte nuclear factor-4α (HNF-4α) in the regulation of the inflammatory response, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokines in a model of knock-down HNF-4α HepG2 cells. The results indicate an extensive role for HNF-4α plays in the regulation of a large number of the liver-specific genes. Majority of genes (71%) affected by cytokine treatment are also affected by HNF-4α knock-down. This significant overlap suggests that HNF-4α may play a role in regulating the cytokine-induced inflammatory response. Overall design: The different treated HepG2 cells were grouped into 4 groups (four replicates in each group): Group1, Control; Group2, HNF-4α shRNA treated cells; Group3, cytokine treated group; Group4, HNF-4α shRNA and cytokine treatments. RNA extraction and hybridization on Affymetrix microarrays were processed. The expression profiles between different groups were analyzed and compared. In order to explore the function of HNF-4α in the inflammatory response, a set of 170 genes annotated as inflammatory response was obtained from GO (geneotology.org), the enrichments of these inflammatory genes were analyzed in different treated groups.
Project description:We developed genetically engineered Hepa/8F5 cells (available from RIKEN BioResource Center [RCB4661]), in which genes of eight liver-enriched transcription factors (LETFs)—hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β [FOXA2], HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ—were transduced into murine hepatoma Hepa1-6 cells as drug-inducible expression cassettes [Biochem. Eng. J., 60, 67–73 (2012)]. Hepa/8F5 cells can induce high liver functions by the addition of an inducer drug (doxycycline; Dox) via overexpression of LETF genes.
Project description:Specific regulation of target genes by transforming growth factor-β (TGF-β) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In the present study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and compared them to those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-β. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells, but not in HaCaT cells, and the HNF4α binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. ChIP-sequencing analysis of HNF4A binding regions under TGF-β stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4A bindings. MIXL1 was identified as a new combinatorial target of HNF4A and Smad2/3, and both the HNF4A protein and its binding motif were required for the induction of MIXL1 by TGF-β in HepG2 cells. These findings generalize the importance of binding of HNF4A on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-β, and suggest that certain transcription factors expressed in a cell-type-specific manner play important roles in the transcription regulated by the TGF-β-Smad signaling pathway. HepG2 cells were treated with TGF-beta for 1.5 h or left untreated. anti-HNF4A ChIP-seq was performed. One lane was used for each sample.
Project description:In order to characterize the differentially expressed miRNAs after the p53 activation , small RNA-seq were used after the overexpression of p53 in HepG2 cells. Four samples of HepG2 cells were subjected to small RNA-seq in two biological replicates.The HepG2 cells were treated with 1µg/ml doxorubicin for 24 hours to induce the expression of p53. The experimental group(dox-treated HepG2：HepG2_24h_rep1 and HepG2_24h_rep2) and control group(untreated HepG2: HepG2_0h_rep1 and HepG2_0h_rep2) were subjected to small RNA-seq to identify the p53-regulated miRNAs.
Project description:Analysis of DDX20 knockdown - hepatocellular carcinoma cells. The expression levels of genes driven by NF-kB and related with carcinogenesis, were significantly enhanced in DDX20-knockdown cells. One condition experiment, HepG2 vs. HepG2-DDX20 knockdown cells
Project description:Viscum coloratum (Kom.) Nakai is a variety of biological activities of medicinal plants, and the active component such as polysaccharides or alkaloids, also proved to have the effect of anti-tumor, anti-virus, anti-radiation, anti-oxidation and anti-infection. In this study, we examined the inhibition of three polysaccharide fractions from Viscum coloratum (Kom.) Nakai on HepG2 cells growth in a dose-dependent manner using CCK-8 kit assay, and flow cytometry analysis showed that VCP2 delayed cell cycle in G1 phase and induced apoptosis in HepG2 cells which may due to overexpressed p21Wafl/Cip1 and Cyclin D and decreased expressions of Cyclin E and CDK4, and the upregulated Bad, Smac and Caspase-3 and the downregulated Bcl-XL and XIAP may be one of reasons for inducing apoptosis in VCP2-treated HepG2 cells, but these effects may be not obviously related to Bad. Using iTRAQ and 2D-LC-MSMS, 113 differentially expressed proteins identified in normal and VCP2-treated HepG2 cells. Among them, the expressions of 59 proteins were upregulation, and the expressions of 54 proteins were downregulation. GO, pathways and PPI of differentially expressed proteins further analyzed in polysaccharides-treated HepG2 cells by bioinformatic approach. These findings widen our understanding the anti-tumor mechanisms of mistletoe polysaccharide and provide new clues for screening potent responsive protein to polysaccharides.
Project description:Increased fatty acid (FA) is often observed in highly proliferative tumors, contributing to promoting proliferation of tumor cells. FA affects secreted factors from tumor cells, which can modulate tumor microenvironment in favor of tumor survival. However, the secreted factors affected by the increased FA have not been systematically explored. Here, we performed comprehensive secretome profiling of oleate-treated and untreated HepG2 cells. Comparison of the secretomes identified 349 differentially secreted proteins (DSPs; 145 up-regulated and 192 down-regulated) in oleate-treated samples, compared to untreated samples. Functional enrichment and network analyses of the DSPs revealed that the 145 up-regulated secreted proteins by oleate treatment were mainly associated with cell proliferation-related processes, such as lipid metabolism, inflammatory response, and ER stress. Based on the network models of the DSPs, we selected four up-regulated secreted proteins (MIF, THBS1, PDIA3, and APOA1) that can represent such processes related to cell proliferation. Thus, our results provided a secretome profile indicative of oleate-induced proliferation of HepG2 cells.
Project description:The microarray results showed that linalool sitimulation of lipid-loaded HepG2 cells rewired the hepatic transcriptome profile, with linalool being comparable to those of fenofibrate. Total of 8,988 genes that were commonly and significantly expressed in all experimental groups, including control hepatocytes, lipid-loaded hepatocytes, and lipid-loaded hepatocyted stimulated with linalool or fenofibrate, respectively, to compare the transcriptome profile of linalool (1mM) with those of hypotriglyceridemic drug, fenofibrate (100 μM). Lipid loading of hepatocytes notably changed hepatic transcriptome profile, where 77 % of the selected genes showed >20% changes in expression (fold change > 1.2 or fold change <0.8). However, linalool stimulation of lipid-loaded cells showed that 48 % of the selected genes had expression changes of >20%. Thus, 29 % of the selected genes became to show less than 20% changes in expression after linalool stimulation compared to the lipid-loaded condition. Fenofibrate showed 46 % of the selected gene expression changes of >20%. Thus, 31 % of the seleted genes fell into the <20% change category compared to the lipid-loaded condition after fenofibrate stimulation. Untreated control vs. lipid-loaded HepG2 cells (3 biological replicates), untreated control vs. lipid-loaded HepG2 cells treated with fenofibrate (3 biological replicates), untreated control vs. lipid-loaded HepG2 cells treated with linalool (2 biological replicates). One replicate per array
Project description:Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1α and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis. We sought to determine the effect of co-exposure of certain drugs associated with idiosyncratic DILI with the cytokine TNFalpha to mimic drug exposure with inflammation signaling in HepG2 cells Human hepatoma HepG2 cells were obtained from American Type Culture Collection (ATCC, Wesel, Germany), cultured in DMEM supplemented with 10% (v/v) FBS, 25 U/mL penicillin and 25 µg/mL streptomycin and used for experiments between passage 5 and 20.
Project description:Investigation of whole genome gene expression level changes in hepatocellular carcinoma cell line hepG2 in regular culture, hepG2-slug in regular culture and hepG2-slug on Matrigel. Whole genome gene expression level changes have been compared in hepatocellular carcinoma cell line hepG2 in regular culture, hepG2-slug in regular culture and hepG2-slug on Matrigel. Roche NimbleGen micro-array analysis was employed to assess global genome expression in HepG2 in regular culture, HepG2-slug in regular culture and HepG2-slug on Matrigel. The results demonstrated that the up-regulated genes and the down-regulated genes increased significantly when HepG2-slug cells with VM forming ablity were cultured on Matrigel and formed VM.