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Project description:Gene disruption is a central tenet of cancer research, where novel drug targets are often identified and validated through cell-growth based knockdown studies or screens. Short hairpin RNA (shRNA) mediated mRNA knockdown is widely used in both academic and pharmaceutical cancer research settings. However, off-target effects of shRNAs as well as interference with endogenous small RNA processing have both been reported. We show here that both gene-specific and control shRNA expression impair in vitro cell growth and preferentially decrease clustered microRNA levels, significantly reducing oncogenic microRNA cluster expression. In addition, exogenous shRNAs induce a depressed cell-cycle-gene expression signature that is shRNA-sequence independent and present across several studies. The collective impact of these observations is important to cancer research, given the widespread historical use of shRNAs in the field and the common goal of interrogating genes that regulate proliferation. We therefore recommend that for future shRNA use, care be taken to titrate lentiviral dose, use hairpin-expressing controls, perform gene-of-interest rescue experiments and/or validate shRNA-derived results by small interfering RNA (siRNA) knockdown or CRISPR/Cas9-mediated genetic knockout.
2025-09-01 | GSE120526 | GEO
Project description:Gene disruption is a central tenet of cancer research, where novel drug targets are often identified and validated through cell-growth based knockdown studies or screens. Short hairpin RNA (shRNA) mediated mRNA knockdown is widely used in both academic and pharmaceutical cancer research settings. However, off-target effects of shRNAs as well as interference with endogenous small RNA processing have both been reported. We show here that both gene-specific and control shRNA expression impair in vitro cell growth and preferentially decrease clustered microRNA levels, significantly reducing oncogenic microRNA cluster expression. In addition, exogenous shRNAs induce a depressed cell-cycle-gene expression signature that is shRNA-sequence independent and present across several studies. The collective impact of these observations is important to cancer research, given the widespread historical use of shRNAs in the field and the common goal of interrogating genes that regulate proliferation. We therefore recommend that for future shRNA use, care be taken to titrate lentiviral dose, use hairpin-expressing controls, perform gene-of-interest rescue experiments and/or validate shRNA-derived results by small interfering RNA (siRNA) knockdown or CRISPR/Cas9-mediated genetic knockout.
2025-09-01 | GSE120525 | GEO
Project description:Short hairpin RNAs artifactually impair cell growth and suppress clustered microRNA expression
| PRJNA493260 | ENA
Project description:Short hairpin RNAs artifactually impair cell growth and suppress clustered microRNA expression [miRNA expression]
| PRJNA493265 | ENA
Project description:Short hairpin RNAs artifactually impair cell growth and suppress clustered microRNA expression [gene expression]
| PRJNA493266 | ENA
Project description:Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting.
| S-EPMC5622367 | biostudies-literature
Project description:BackgroundEffective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.ResultsWe have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.ConclusionWe demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.
| S-EPMC2194719 | biostudies-literature
Project description:The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibits significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based model with high predictive power. Using this model, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and transformed them into agshRNAs. We validated the performance of our agshRNA design by evaluating antiviral efficacies of agshRNAs in cells infected with different viruses. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RNA-induced silencing complex (RISC). We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develops a novel antiviral strategy that combines machine learning-based algorithm with agshRNA design to custom design antiviral agshRNAs with high efficiency.
| S-EPMC11401469 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2020-12-31 | GSE156880 | GEO
Project description:In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology. Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate approximately 21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila.
| S-EPMC2735555 | biostudies-literature