RNAi screen in tumor cells identifies artificial microRNAs that improve oncolytic virus replication
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE291763 | GEO | 2025/05/09
REPOSITORIES: GEO
Similar Datasets
Project description:RNAi screen in tumor cells identifies artificial microRNAs that improve oncolytic virus replication
| PRJNA1235208 | ENA
Project description:To allow exclusion of non-expressed genes from the hit identification process, microarray analyses of (i) Allstars transfected and Chikungunya (CHIKV) infected, (ii) Allstars transfected and non-infected, (iii) non-transfected and CHIKV infected and (iv) non-transfected and non-infected 293 cells were performed. Transfection was done by adding 4x10e5 293 cells (in complete DMEM medium) to a mixture of serum-free RPMI medium, HiperFect (Qiagen) and Allstars siRNAs (Qiagen, final siRNA concentration: 20 nM). Infection with CHIKV-GFP was performed 72 h p.t. at an MOI of ~3. Cells were lysed 18 h p.i. using Trizol (Invitrogen), followed by RNA isolation, that was subsequently subjected to microarray analysis.
2016-05-09 | GSE69980 | GEO
Project description:Proper DNA replication is critical to maintain genome stability. When the DNA replication machinery encounters obstacles to replication, replication forks stall and the replication stress response is activated. This response includes activation of cell cycle checkpoints, stabilization of the replication fork, and DNA damage repair and tolerance mechanisms. Defects in the replication stress response can result in alterations to the DNA sequence causing changes in protein function and expression, ultimately leading to disease states such as cancer. To identify additional genes that control the replication stress response, we performed a three-parameter, high content, whole genome siRNA screen measuring DNA replication before and after a challenge with replication stress as well as a marker of checkpoint kinase signalling. We identified over 200 replication stress response genes and subsequently analyzed how they influence cellular viability in response to replication stress. These data will serve as a useful resource for understanding the replication stress response.
| S-EPMC4651756 | biostudies-literature
Project description:All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.
| S-EPMC2574945 | biostudies-literature
Project description:Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.
| S-EPMC5071646 | biostudies-literature
Project description:Venezuelan equine encephalitis virus is a member of the alphavirus family and genus togaviridae. VEEV is highly infectious in aerosol form and has been weaponized in the past making it a potential biothreat agent. At present, there are no FDA approved antiviral treatments or vaccines for VEEV. Artificial microRNAs are small molecules which are expressed through endogenous microRNA machinery by RNA polymerase II. These artificial microRNAs effectively inhibit gene expression and are non-toxic to the host cell. VEEV RNA dependent RNA polymerase (RdRp) is central to VEEV replication. Therefore, we hypothesize that targeted inhibition of VEEV RdRp using artificial microRNAs may efficiently inhibit VEEV replication. Five artificial microRNAs were tested in vitro in BHK cells. Three of these artificial miRNAs showed significant inhibition of VEEV replication. Further, these microRNAs were cloned into the expression vector in combination to see the synergistic effect on VEEV replication. Combination of more than one miRNA did not result in significant inhibition of virus replication. In conclusion, we have shown that RNAi through artificial microRNAs effectively inhibits VEEV replication and is significantly less toxic in comparison to siRNAs.
| S-EPMC7113778 | biostudies-literature
Project description:In addition to silencing intended target genes, transfected siRNAs regulate numerous unintended transcripts through a mechanism in which the equivalent of a microRNA-like seed region in the siRNA recognizes complementary sequences in transcript 3' UTRs. The ability to limit such off-target effects would substantially facilitate accurate interpretation of RNA interference (RNAi) experiments and thus greatly enhance their value. We tested whether lentivirus-mediated delivery of shRNA is prone to the seed region-based off-target activity prevalent in siRNA experiments. We compared target gene silencing and overall impact on global gene expression caused by multiple 21-mer duplex sequences delivered as both transfected siRNA and lentivirus vector-expressed shRNA. At equivalent levels of target gene silencing, signatures induced by shRNAs were significantly smaller than those induced by cognate siRNAs and arose less frequently from seed region activity. Interestingly, the low level of seed-region based off-target activity exhibited by shRNAs resulted in down-regulation of transcripts that were largely distinct from those regulated by siRNAs. On the basis of these observations, we recommend lentivirus-mediated RNA interference for pathway profiling experiments that measure whole genome transcriptional readouts as well as for large-scale screens when resources for extensive follow up are limited.
2010-06-09 | GSE22209 | GEO
Project description:BackgroundRNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs), causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s).ResultsWe identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects) is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death.ConclusionsWe have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.
| S-EPMC2996961 | biostudies-literature
Project description:Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1alpha(-/-) murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1alpha, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.
| S-EPMC2453327 | biostudies-literature
Project description:New therapies are needed for metastatic breast cancer patients. Oncolytic herpes simplex virus (oHSV) is an exciting therapy being developed for use against aggressive tumors and established metastases. Although oHSV have been demonstrated safe in clinical trials, a lack of sufficient potency has slowed the clinical application of this approach. We utilized histone deacetylase (HDAC) inhibitors, which have been noted to impair the innate antiviral response and improve gene transcription from viral vectors, to enhance the replication of oHSV in breast cancer cells. A panel of chemically diverse HDAC inhibitors were tested at three different doses (<, = , and >LD50) for their ability to modulate the replication of oHSV in breast cancer cells. Several of the tested HDAC inhibitors enhanced oHSV replication at low multiplicity of infection (MOI) following pre-treatment of the metastatic breast cancer cell line MDA-MB-231 and the oHSV-resistant cell line 4T1, but not in the normal breast epithelial cell line MCF10A. Inhibitors of class I HDACs, including pan-selective compounds, were more effective for increasing oHSV replication compared to inhibitors that selectively target class II HDACs. These studies demonstrate that select HDAC inhibitors increase oHSV replication in breast cancer cells and provides support for pre-clinical evaluation of this combination strategy.
| S-EPMC3961437 | biostudies-literature