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Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells.

Abstract
Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.
AuthorsTimothy R Wagenaar, Tatiana Tolstykh, Chaomei Shi, Lan Jiang, JingXin Zhang, Zhifang Li, Qunyan Yu, Hui Qu, Fangxian Sun, Hui Cao, Jack Pollard, Shujia Dai, Qiang Gao, Bailin Zhang, Heike Arlt, May Cindhuchao, Dietmar Hoffmann, Madelyn Light, Karin Jensen, Joern Hopke, Richard Newcombe, Carlos Garcia-Echeverria, Christopher Winter, Sonya Zabludoff, Dmitri Wiederschain
JournalNucleic acids research (Nucleic Acids Res) Vol. 43 Issue 2 Pg. 1204-15 (Jan 2015) ISSN: 1362-4962 [Electronic] England
PMID25550434 (Publication Type: Journal Article)
Copyright© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
Chemical References
  • DNA-Binding Proteins
  • Endosomal Sorting Complexes Required for Transport
  • MIRN21 microRNA, human
  • MicroRNAs
  • Oligonucleotides
  • Transcription Factors
  • Tsg101 protein
Topics
  • Animals
  • Biological Transport
  • Cell Line, Tumor
  • DNA-Binding Proteins (physiology)
  • Endosomal Sorting Complexes Required for Transport (antagonists & inhibitors, metabolism, physiology)
  • Female
  • Humans
  • Mice, SCID
  • MicroRNAs (antagonists & inhibitors, metabolism)
  • Neoplasms (genetics, metabolism)
  • Oligonucleotides (metabolism)
  • Transcription Factors (physiology)

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