Genome-wide microRNA-expression analysis after p53 activation in hepatocellular carcinoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE74588 | GEO | 2015/11/03
SECONDARY ACCESSION(S): PRJNA300875
REPOSITORIES: GEO
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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 HepG2ï¼HepG2_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.
2015-11-03 | E-GEOD-74588 | biostudies-arrayexpress
Project description:In order to characterize the differentially expressed mRNAs after the p53 activation ,RNA-seq were used after the overexpression of p53 in HepG2 cells.
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Project description:Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. MEG3 functions as a tumor suppressor in hepatoma cells, whose action is mediated by interaction with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes. Total RNA was isolated from hepatoma cells (HepG2 and SK-Hep-1) transfected with either control or MEG3 overexpression/knockdown constructs for global expression analysis.
2015-06-17 | E-GEOD-69916 | biostudies-arrayexpress
Project description:In order to obtain a global picture regarding regulation of p53 in liver cells we used HepG2 hepatoma cells.We created two isogenic sub-cultures of HepG2 cells with altered expression of p53. HepG2 cells were infected with a retroviral vector harboring either small-hairpin RNA targeting p53 (termed HepG2sh-p53) or a non relevant small-hairpin RNA (termed HepG2sh-con). Cells were treated with two different p53 stimulators - Nutlin and AICAR
2012-03-30 | E-GEOD-30137 | biostudies-arrayexpress
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3â??-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.
2016-05-24 | MSV000079767 | MassIVE
Project description:Cancer cells voraciously consume nutrients to support their growth, exposing a metabolic vulnerability that can be therapeutically exploited. Here we show in hepatocellular carcinoma (HCC) cells, xenografts, and in patient-derived HCC organoids that fasting can synergistically sensitise resistant HCC to sorafenib. Mechanistically, sorafenib acts non-canonically as inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR-signalling, prevents this Warburg shift. p53 is necessary and sufficient for the sorafenib-sensitizing effect of nutrient restriction and crucial for improvement of sorafenib efficacy through intermittent fasting (IF) in an orthotopic HCC mouse model. Together, our data indicate IF and sorafenib as clinically actionable, rational combination therapy for HCC with intact p53 signalling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies with the goal of improving therapy response in advanced-stage, and possibly even early-stage, HCC.
2022-02-16 | PXD022723 | Pride
Project description:Development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. We have previously demonstrated that constitutively photomorphogenic 1 (COP1), which regulates p53 activity by ubiquitination, is frequently overexpressed in human HCC. Here we examined whether molecular targeting of COP1 by small interfering (si) RNA can affect the course of HCC progression. COP1-1 was selected as the most effective target siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Interestingly, the growth inhibition occurred both in HCC cells that retain wild-type p53 or express mutant p53 (Y220C or R249S). Next we have determined to investigate the molecular mechanisms underlying the phenotypic changes. Given the recent findings that COP1 functions as a negative regulator of p53, we addressed whether the phenotypic changes caused by COP1 silencing were due to alterations in p53 and/or p21 status. Indeed, in COP1-depleted HepG2 cells expressing wild type p53, induction of apoptosis was associated with restoration of p53 function as judged by a marked increase in the levels of p53 and its target p21, suggesting that cell death was p53-dependent. However, the COP1 silencing in Huh7 cells, which carry Y220C mutation, caused a strong induction of apoptosis without changing p53 levels. To further address this issue, we next looked for the global transcriptional changes underlying the antitumor effects of COP1 silencing in HCC cells [with different p53 status]. For this purpose, Huh7 and HepG2 were treated with NCsiRNA and COP1-1siRNA for 48 hours and subjected to illumina microarray analysis. We found that the number of differentially expressed genes which displayed more than a 2-fold change (P < 0.01 by Bootstrap t-test) was 462 (167 up- and 295 down-regulated genes) and 522 (179 up- and 343 down-regulated genes) in COP1siRNA-treated Huh7 and HepG2 cells, respectively. As expected, the expression levels of RFWD2 (COP1) mRNA were significantly reduced in both treated HCC cell lines. Consistent with phenotypic changes, COP1-depleted Huh7 cells also displayed changes in p53-associated group of genes functionally involved in regulation of apoptosis, growth and differentiation including CASP6, GLIPR1, FHL2, GADD45A, HMOX1 BCL6, FOXO3, GDF15, and SCNN1A genes. Finally, we generated the common COP1 knockdown gene signature which included 78 genes. The Ingenuity Pathway Analysis analysis revealed that the 5 top putative networks with high score (>19) were strongly associated with NF-κB, HNF4α, TNF, and p53 pathways, suggesting that a common subset of molecular alterations in the diverse oncogenic pathways may cooperatively result in growth inhibition of HCC cells with wild type and mutant p53. Analysis of COP1 knockdown gene expression signatures by microarray revealed that the anti-proliferative effect was driven by a common subset of molecular alterations including p53-associated functional network. Systemic delivery of a modified COP1siRNA by stable-nucleic-acid-lipid-particles (SNALP) significantly suppressed neoplastic growth in liver, without unwanted immune response in an orthotopic xenograft model. These findings provide the first proof of principle that COP1 is a promising target for systemic therapy of HCC. Microarray was performed on human Ref-8v3 microarrays (illumina) as recommended by the manufacture. In detail, total RNAs were isolated 48 h after the transfection of NCsiRNA or COP1-1siRNA to Huh7, HepG2 or Hep3B cells. Biotin-labeled cRNA was linearly amplified according to manufacturerâs specification (AMIL1791; Ambion, Austin, TX). As input, 200 ng total RNA from tumor was used for the in vitro transcription (IVT) reactions which were incubated for 16 h at 37 ºC. The efficiency of this single round amplification was measured by NanoDrop (ND1000, Thermo Scientific). Hybridization, washing, detection (Cy3-streptavidin, Amersham Biosciences, GE Healthcare) and scanning were performed on an Illumina iScan system (Illumina) using reagents and following protocols supplied by the manufacturer. Briefly, the biotinylated cRNA (750 ng/sample) was hybridized on Sentrix whole genome beadchips human Ref-8v3 for 18 h at 58ºC while rocking (5 rpm). The beadchip covers ~ 24,000 RefSeq transcripts. Image analysis and data extraction were performed automatically using Illumina GenomeScan Software. To explore the functional relationships among the genes with altered expression in the HCC cells treated with COP1-1siRNA, a pathway analysis was carried out with the Ingenuity Pathway Analysis tool (Ingenuity Systems). Using the approach, we examined functional associations among genes and generated the gene networks with high significance on the basis that they had more of the interconnected genes present than would be expected by chance. The significance of each network was estimated by scoring system provided by Ingenuity. The scores are determined by the number of differentially expressed genes within each of the networks and the strength of the associations among network members. Once over-represented genes that are functionally relevant in gene networks are identified, we validated their functional association by using the independent pathway analysis tool PathwayStudio (Ariadne Genomics).
2010-11-01 | E-GEOD-21955 | biostudies-arrayexpress
Project description:Genome-wide RNA-expression analysis after p53 activation in hepatocellular carcinoma
| PRJNA634530 | ENA
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3’-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.
2015-07-23 | PXD001976 | Pride