RNA-seq of H1299 cells stably transfected with empty vector or the p53 mutant R273H.
ABSTRACT: This experiment aimed to determine the genes that were upregulated when mutant p53 (R273H) was expressed. We used H1299 lung cancer cells that are endogenously p53-null and overexpressed the p53 mutant.
Project description:This experiment was performed to determine which gene promoters mutant p53 binds and transcriptionally regulates in order to understand how mutant p53 accomplishes its gain of function phenotype.
Project description:In the present study, we used a high-throughput small RNA deep sequencing followed by a systematic computational analysis to identify genome wide mutant p53R273H regulated miRNAs in both DNA damage dependent and independent context. Several miRNA-mRNA regulatory networks have been predicted that might contribute to mutant p53 GOF properties. Differentially regulated miRNA signature profile has been validated in the lung cancer patients harboring wildtype and mutant p53. We identified specific miRNA signatures for lymph node metastasis associated with p53 mutation in lung adenocarcinoma and also predicted the possible contribution of two mutant p53 regulated miRNAs in EMT process. Furthermore, this study identified a hitherto unknown miRNA in human which might act as one of the crucial downstream targets of GOF mutant p53 to confer oncogenic properties. Determination of mutant p53R273H regulated microRNAs H1299 cells.
Project description:Transcriptional profiling of H1299 non-small cell lung carcinoma cells transfected with either wt p53 or mut(175) p53 driven by the 5xHRE promoter (5 repeats of hypoxia-inducible factor response elements) and treated for 16 h with normoxia (21% O2) or hypoxia(<0.1% O2). 5xHRE promoter ensures that p53 expression is induced in hypoxic conditions only. Goal was to determine the transcriptional response of p53 in hypoxia and the 175 p53 mutant was used as a control as it is DNA-binding defective and transcription-incompetent mutant. Four-condition experiment: wt p53-transfected H1299 cells treated with normoxia, mut p53-transfected H1299 cells treated with normoxia, wt p53-transfected H1299 cells treated with hypoxia, mut p53-transfected H1299 cells treated with hypoxia. Biological replicates: 1 normoxic sample with wt p53, 1 normoxic sample with mut p53, 3 hypoxic samples with wt p53, 3 hypoxic samples with mut p53.
Project description:The human TP53 gene is frequently mutated in tumors and cell lines. Unlike other tumor suppressors that are commonly inactivated by deletions or nonsense mutations, the majority of p53-mutations are missense point mutations that result in the expression of a full-length protein with an altered amino acid that has lost sequence specific DNA-binding. Expression of mutant p53 (mutp53) confers advantages to tumor cells and transcriptional regulation of several genes mediating the beneficial effects has been shown to play a role. However, molecular mechanisms of transcriptional regulation by mutp53 are still poorly understood. We used the glioblastoma-derived U-251 MG human cell line endogenously expressing mutp53 protein (R273H mutation) to analyze gene expression profiles on Agilent Whole Human Genome Microarray after transient and stable depletion of mutp53 expression. Gene expression data was correlated with a ChIP study on a custom tiling array to understand the contribution of endogenously expressed mutp53 to transcriptional regulation. This series of microarray experiments contains the gene expression profiles of glioblastoma-derived U-251 MG human cell lines engineered to constitutively express a p53-specific shRNA or scrambled control shRNA. To reverse the effect of mutp53 depletion, stable clones were modified by stable integration of a mutp53-R273H expression construct or empty pCDNA3 vector as a control. In addition, we performed gene expression analysis of U-251 MG cells transiently transfected with p53-specific siRNA or control siRNA (3 biological replicates each).
Project description:To evaluate the effect on gene expression by p53 family members and AKR1B10, we overexpressed p53 family members in H1299 cells or knocked down AKR1B10 in HCT116 cells and evaluated the gene expression by microarray analysis. Gene expression was measured in H1299 cells infected with adenovirus expressing p53, p63g and p73b, and in HCT116 cells transfected with siRNAs targeting AKR1B10 and then treated with adriamycin.
Project description:Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin β1 and down- stream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients. We performed cDNA microarray analysis to compare two stably expressing cell lines originating from SNB19; two clones expressing a mutant form of IGFBP2 that cannot bind integrin (RGD → RGE point mutation; referred to as RGE mutant); and two clones expressing wild-type IGFBP2. SNB19 clones transfected with empty vector were placed in the reference channel in each hybridization.
Project description:H1299 cells were overexpressed miR-138 or silenced AGO2. The expression of 92 genes associated with p53 using the “Human p53 Signaling Pathway PCR Array” qPCR gene expression profiling. H1299 cells were transfected with NC mimics, AGO2 siRNA or miR-138 for 48h. Equal amount total RNA from each group was pooled prior to gene expression analysis.
Project description:To evaluate the effect on lincRNA expression by p53 family members, we overexpressed p53 family members in H1299 cells and evaluated the lincRNA expression by microarray analysis. lincRNA expression was measured in H1299 cells infected with adenovirus expressing LacZ, p53, p63a, p63g, p73a and p73b.