Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4+CD25+ T regulatory cells activation
Ontology highlight
ABSTRACT: We recently described the phenotype of HepG2 and Huh-7, hepatocellular carcinoma cells, knocked down for histone variant macroH2A1. Both cell lines acquire a cancer stem cell phenotype (Lo Re O et al., Hepatology 2017, PMID: 28913935; Lo Re O et al., Epigenetics 2018, PMID: 30165787). We found that short hairpin RNA-mediated macroH2A1 knockdown induced acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolytic pathways. As macroH2A1 is a potent transcriptional modifier we asked how KD of this histone variant might affect patterns of gene expression, and whether we could identify potential mechanistic links to the observed in vitro and in vivo HCC phenotypes. Here we used RNA-Seq to gain deep mechanistic insight into the distinct functions of macroH2A1 KD and conditioned medium (CM)-exposed Huh-7 cells. Heatmap analysis of the differentially expressed genes between the three groups revealed a similar transcriptomic profile between KD and CM, compared to the control condition. Genes were considered differentially expressed between groups if their expression values significantly differed by >2 fold. Statistical differences in gene expression were assessed by the ANOVA test. Correction for multiple test was achieved by the Benjamini-Hochberg procedure. The significance threshold was set to 0.05. Using a cut-off of 2 fold change, assessment of differentially expressed genes for Huh-7 macroH2A1 KD or Huh-7 CM KD versus CTL showed no transcriptional overlap between the different Huh-7 cell lines. 783 and 987 genes were instead significantly differentially-expressed in macroH2A1 KD or CM KD versus control cells, respectively. Interestingly, the significantly enriched transcripts over-represented in KD and CM-exposed cells compared to control cells were implicated in a number of functions and diseases that were also shared between CM versus control comparisons. Specifically, Ingenuity pathway analysis (IPA) identified categories of cancer, gastrointestinal diseases, lipid metabolism, cell-to-cell signaling, nucleic acid metabolism, cell death & survival and others, in common between KD versus CTL and CM versus CTL. These data support a paracrine modulation of gene expression by macroH2A1 KD in HCC cells.
Project description:We recently described the phenotype of HepG2 and Huh-7 hepatocellular carcinoma cells deleted for histone variant macroH2A1, which acquire cancer stem cell phenotype (Lo Re O et al., Hepatology 2017, PMID: 28913935). We found that short hairpin RNA-mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. As macroH2A1 is a potent transcriptional modifier we asked how KD of this histone variant might affect patterns of gene expression, and whether we could identify potential mechanistic links to the observed in vitro and in vivo HCC phenotypes. We obtained and validated an RNA-Seq dataset (Borghesan M et al., Cancer Res 2016, PMID: 26772755; Lo Re O et al., Hepatology 2017, PMID: 28913935), to conduct an unbiased comparison of the transcriptional profiles of HepG2 macroH2A1 KD cells and controls. Using a cut-off of 2 fold change, assessment of differentially expressed genes for macroH2A1 KD versus CTL showed no transcriptional overlap between the different HepG2 cell lines. Considering all differentially-expressed genes, there was significant enrichment (–log(p-value) > 1.3) in several functions and pathways that regulate pluripotency of human embryonic stem cells.
Project description:To clarify the molecular mechanism by which FNDC4 promotes HCC invasion, mass spectrometry (MS) analysis was performed on the lysates of Huh-7-overexpressing FNDC4 and normal Huh-7 cells.
Project description:HCC cell line, Huh-7 cells and HCC-LM3 cells, was transfected with METTL5 sgRNA to knockout METTL5 expression., and check the downstream mRNA changes.
Project description:Small nucleolar RNAs (snoRNAs) dysfunction have been associated with cancer development. We investigated the function of an orphan C/D box class snoRNA, SNORD126, in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cells We used microarrays to identify targets with roles in SNORD126âs activity in Huh-7 cells SNORD126- or EGFP-overexpressed Huh-7 cells were collected and followed by RNA extraction, then hybridized with Affymetrix microarrays. We sought to obtain the differentially expressed genes between the two groups.
Project description:Analysis of HSF1-down regulation of HCC cells at gene expression level. Results provide important information of genes involved in HSF1, such as liver proliferation, apoptosis, stress response, metabolism, these genes were up- or down-regulated. Total RNA obtained from HSF1-KD KYN2 (HCC) cells and HSF1-control KYN2 cells. To identify genes generally involved in HSF1 associated, we compared expression profiles between HSF1-KD KYN2 (HCC) cells and HSF1-control KYN2 cells by using Illumina HumanWG-6 BeadChip.
Project description:The study aims to assess the influence of soluble factors from HCC on MSCs and to investigate the regulatory effect of miRNAs in human adipose- MSCs (hA-MSCs) cultured in HCC microenvironment.The results suggest an interaction between tumor cells and the surrounding stromal components and provide evidence to the direct generation of CAF phenotype upon exposure of hA-MSCs to Huh-7 cancer microenvironment, favoring cancer progression. This might shed the light on the fate of hA-MSCs use in therapy in HCC. hA-MSCs modulation may be achieved via dysregulation of miR17-5P and 615-5p expression. These data suggest that miRNAs may present a new target for HCC treatment.
Project description:HCC cell line, Huh-7 cells and HCC-LM3 cells, was transfected with sh-WDR4-2 or sh-NC for 48hr to knockdown WDR4 expression, and check the downstream mRNA changes.
Project description:Metformin is a kind of widely used antidiabetic agent, which regulates glucose homeostasis through inhibiting liver glucose production and increasing glucose uptake in muscle. Recent studies suggest that metformin exhibits anticancer properties in a variety of cancers. Although several antitumor mechanisms have been proposed for metformin action, its mode of action in human liver cancer remains not elucidated. In our study we investigated the underlying molecular mechanisms of metformin’s antitumor effect on Huh-7 cells of hepatocellular carcinoma (HCC) in vitro. RNA sequencing (RNA-seq) was performed to explore the effect of metformin on the transcriptome of Huh-7 cells. The results revealed that 4518 genes (with log2 fold change>1 or < -1, p-adjusted value<0.05) were differentially expressed in Huh-7 cells with treatment of 25mM metformin compared to 0mM metformin including 1812 up-regulated and 2706 down-regulated genes. Gene ontology and KEGG pathway analyses identified 54 classical pathways which were significantly enriched, and 16 pathways are closely associated with cancer, such as cell cycle, DNA replication, ECM-receptor interaction and so on. We selected 11 differentially expressed genes, which are closely associated with HCC to validate their differential expressions through quantitative real-time reverse transcription PCR (qRT-PCR). The result exhibited that the genes of FASN, MCM6, MCM5, MARCKS, FADS2, CXCL1, BMP4, SKP2, KNG1, PCNA were down-regulated and DUSP1 is significantly up-regulated in Huh-7 cells with treatment of 25mM metformin. These differentially expressed genes and pathways might play a crucial part in the antitumor effect of metformin, and might be potential targets of metformin treating HCC. Further investigations are required to evaluate the metformin mechanisms of anti-cancer action in vivo.
Project description:To investigate the inhibitory effect of K458R mutation of HNF4A on HCC, we established Huh-7 xenografts treated with Ad-GFP, Ad-HNF4A or Ad-K458R. We then performed gene expression profiling analysis using data obtained from RNA-seq of three groups of Huh-7 xenografts treated with adenovirus.
Project description:To globally analyse the lncRNAs with potential coding ability of HCC cells, we performed Ribo-seq using HuH-7 cells. The cycloheximide (CHX) was added to inhibit the translational elongation of ribosomes of the HuH-7 cells.Furthermore,we identified the candidate ORFs with the selected RPF reads using the RiboCode software.