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Short hairpin RNAs artifactually impair cell growth and suppress clustered microRNA expression [miRNA expression]

ABSTRACT: 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.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE120526 | GEO | 2025/09/01

REPOSITORIES: GEO

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Short hairpin RNAs artifactually impair cell growth and suppress clustered microRNA expression [gene expression]

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.

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A genome-scale shRNA resource for transgenic RNAi in Drosophila

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An optimized microRNA scaffold increases shRNA processing and target knockdown

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A genome-scale shRNA resource for transgenic RNAi in Drosophila

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iTRAQ-based quantitative proteomic analysis of nuclear proteome in VPS35-depleted HeLa cells

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Regulatory RNA Apela Is a Component of a Negative Feedback Loop Regulating p53-Mediated Apoptosis in Embryonic Stem Cells (I)

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Small and large RNA sequencing of mouse livers receiving small hairpin RNAs

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