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Cited by 534MicroRNA in disease regulationRNA Interference and Gene DeliveryPlant Molecular Biology Research

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

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Khan, Aly A, Betel, Doron, Miller, Martin L, Sander, Chris, Leslie, Christina S, Marks, Debora S·2009·Nature Biotechnology
DOI 10.1038/nbt.1543PMC PMC2782465
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Abstract

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

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  • When multiple miRNAs are packaged within a single construct, they may compete for processing by the cellular miRNA biogenesis machinery, leading to reduced efficiency in individual miRNA maturation and function.

    “Consistent with this possibility, when multiple miRNAs are packaged within a single construct, they may compete for processing by the cellular miRNA biogenesis machinery, leading to reduced efficiency in individual miRNA maturation and function 44,45 .”

    From rAAV-mediated Multi-miRNA Delivery Targeting MAPK Pathway Suppresses Laser-induced Choroidal Neovascularization in Mice (2026) · cites this paper as [45]

    The paper’s Abstract (page not available) proposes that transfected small RNAs compete with endogenous miRNAs for RISC or other downstream machinery, reducing endogenous miRNA function. It does not establish that multiple miRNAs packaged in one construct compete during processing or that their individual maturation is less efficient. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • The high level of miRNA overexpression may not represent native miRNA targeting, as recent studies suggest limited abundance of AGO2 may drive competition between miRNAs for proper loading and function.

    “the high level of over-expression may not represent native miRNA targeting as recent studies have suggested that limited abundance of AGO2 itself may drive competition between miRNAs for proper loading and function (Khan et al., 2009;Tan et al., 2019).”

    From Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP (2022) · cites this paper as Khan et al., 2009

    The paper proposes that transfected small RNAs compete with endogenous miRNAs for RISC or related machinery, and its Discussion warns that this can affect experiments used to assess miRNA targets (pages unavailable in our copy). It does not establish that AGO2 specifically is limiting or that competition for AGO2 loading is the mechanism; the Discussion says the precise mechanism remains unresolved. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

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Authors
Khan, A. A., Betel, D., Miller, M. L., Sander, C., Leslie, C. S., & Marks, D. S.
Affiliations
  • Columbia University (US)
  • Memorial Sloan Kettering Cancer Center (US)
  • University of Copenhagen (DK)
  • Harvard University (US)
Corresponding author
  • Leslie, Christina S
  • Marks, Debora S
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2009-05-24
Volume
27
Issue
6
Pages
549-555
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/nbt.1543
PMC
PMC2782465
OpenAlex
W2020469175
Cited by
534
References
6
Citations by year
2026: 15 · 2025: 21 · 2024: 24 · 2023: 21 · 2022: 27 · 2021: 21 · 2020: 27 · 2019: 24 · 2018: 29 · 2017: 25 · 2016: 28 · 2015: 41 · 2014: 44 · 2013: 46 · 2012: 48
FWCI
15.77
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (submittedVersion)
Funders
  • National Institutes of Health (5r01ca124380-04)
  • National Cancer Institute (U54CA121852, CA121852., CA124380)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Keywords
transfection, siRNA, microRNA, endogenous microRNAs, gene upregulation, saturation effects, RNA interference, gene expression profiling, miRNA target prediction, siRNA design, shRNA screen, siRNA therapeutics
MeSH terms
Cell Line, Gene Expression Regulation, Humans, Models, Genetic, Transfection, Up-Regulation, Statistics, Nonparametric, Genes, cdc, Gene Expression Profiling, RNA, Small Interfering, RNA-Induced Silencing Complex, MicroRNAs, Cell Line, Tumor, Gene Knockdown Techniques

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