Project description:To have a global picture of the miRNAs dependent on the transcription factor E2F1, we assessed small RNA changes, by RNA-sequencing, of HeLa cells transfected with an siRNA targeting E2F1 or a control siRNA.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are using RNA sequencing analysis to evaluate the effects of si-E2F1 (or E2F3) on the mRNA of human nasopharyngeal carcinoma cell line HK1. Methods: Human nasopharyngeal carcinoma cell line HK1 was transfected with a control non-targeting siRNA to cells or transfected with siRNA targeting E2F1 (or E2F3) for 48 hours in DMEM medium (with 10% serum). Total RNA were extracted and detected by Illumina high-throughput RNA sequencing data analysis. 3 independent biological replicates were plated, transfected in parallel for each control and siRNA. Results: Log-fold changes of up- or down-regulated mRNAs between the control and experiment group were selected with a significance threshold of p<0.05. Conclusions: Our study describes the mRNA changes of human nasopharyngeal carcinoma cell line HK1 transfected with E2F1 (or E2F3) siRNA.
Project description:This SuperSeries is composed of the following subset Series: GSE32353: Expression data from E2f7/E2f8 and E2f1/E2f2/E2f3 null and wild type liver along with E2f7/E2f8 null and wild type trophoblast giant cells (nCounter) GSE32354: Expression data from E2f7/E2f8 and E2f1/E2f2/E2f3 null liver (Affymetrix) Refer to individual Series
Project description:To investigate mechanism involved in the development of NAFLD-driven hepatocarcinoma (HCC), we used genome microarray expression profiling. The aim was to identify genes which upregulation could be controlled by E2F1 and/or E2F2 in NAFLD -driven HCC. For that, HCC was induced in wild type (WT), E2f1 knockout mice (E2f1-/-) and E2f2 knockout mice (E2f2-/-) by administration of the procarcinogenic agent diethylnitrosamine (DEN) combined with a feeding of high fat diet (HFD) for 32 weeks.The microarray results were then validated by RT-qPCR. For that, the expression of the selected genes (some overexpressed, some downregulated and others with no change) was measured in the same RNA samples by real time PCR. The results were clustered in pathways with the Reactome Database.
Project description:The E2F family consists of transcriptional repressors and activators that control cell proliferation. In the classic paradigm of cell cycle regulation, the three activators, E2F1, E2F2 and E2F3, are invariably depicted as the final components of a CDK/Rb signaling cascade that executes the transcriptional program necessary to commit cells to enter S phase. Unexpectedly, we find through analysis of Affymetrix expression array data that mature lens epithelial cells deficient for E2F1-3 fail to repress cell cycle-regulated genes (and other targets of E2F) and that this corresponds with subsequent apoptosis and cellular collapse in the lens. Murine lenses were collected at two stages of development for RNA extraction and hybridization on Affymetrix microarrays. Our aim was to determine key events that lead to cellular collapse of lenses triply deficient for E2F1, E2F2, and E2F3 in neonates.
Project description:The E2F family consists of transcriptional repressors and activators that control cell proliferation. In the classic paradigm of cell cycle regulation, the three activators, E2F1, E2F2 and E2F3, are invariably depicted as the final components of a CDK/Rb signaling cascade that executes the transcriptional program necessary to commit cells to enter S phase. Unexpectedly, we find through analysis of Affymetrix expression array data that mature lens epithelial cells deficient for E2F1-3 fail to repress cell cycle-regulated genes (and other targets of E2F) and that this corresponds with subsequent apoptosis and cellular collapse in the lens.
Project description:Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended (“off-target”) consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3’ UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence-mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with similar dependence on siRNA concentration as silencing of the target transcript. These results demonstrate that short stretches of sequence complementarity to the seed region of the siRNA are key to the silencing of unintended transcripts, and that this limits the specificity of siRNA-mediated transcript silencing. Because these off-target events are sequence-dependent, inclusion of multiple independent siRNAs to the target of interest can help to distinguish true positives from false positives in functional genetic analyses. Keywords: siRNA, RNAi, sequence alignment, off-target, seed region