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Nucleic Acids Res. 2015 Jan;43(2):1204-15. doi: 10.1093/nar/gku1367. Epub 2014 Dec 30.

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

Author information

1
Sanofi Oncology, Cambridge, MA 02139, USA.
2
Regulus Therapeutics, San Diego, CA 92121, USA.
3
Sanofi Oncology, Cambridge, MA 02139, USA dmitri.wiederschain@sanofi.com.

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.

PMID:
25550434
PMCID:
PMC4333411
DOI:
10.1093/nar/gku1367
[Indexed for MEDLINE]
Free PMC Article

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