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.
Highlights
MicroRNAs are a class of evolutionarily conserved, short non-coding RNAs that play a critical regulatory role in many processes, including control of cellular development, metabolism, cell cycle and apoptosis. miRNAs exert their function through post-transcriptional regulation of mRNA stability or inhibition of translation through an interaction with the 3 UTR [1]
No increase in luciferase activity was observed upon treatment with MM control, indicating this assay was specific for miR-21 inhibition as opposed to non-specific effects associated with transfection of short oligonucleotides
Knockdown of either tumor susceptibility gene 101 (TSG101) or VPS28 restored productive free uptake in both resistant CLONE9 and CLONE15 (Figure 3E). These results indicate that the loss of productive uptake in SKHEP1 can be overcome by the knockdown of the ESCRT-I components, and adds further evidence for the role of ESCRT-I in the regulation of productive transport of anti-miR-21 into cancer cells
Summary
MicroRNAs (miRNAs or miRs) are a class of evolutionarily conserved, short non-coding RNAs that play a critical regulatory role in many processes, including control of cellular development, metabolism, cell cycle and apoptosis. miRNAs exert their function through post-transcriptional regulation of mRNA stability or inhibition of translation through an interaction with the 3 UTR [1]. MicroRNAs (miRNAs or miRs) are a class of evolutionarily conserved, short non-coding RNAs that play a critical regulatory role in many processes, including control of cellular development, metabolism, cell cycle and apoptosis. Accumulating evidence suggests that loss of function or overexpression of miRNAs contributes to the development and progression of many common human diseases, including metabolic syndromes, heart disease and cancer [2]. Numerous studies have demonstrated miR-21 to be upregulated in a wide range of human cancers, and elevated miR-21 levels are consistently associated with poor patient prognosis [4,5]. Given its prominent role in human disease, therapeutic utility of miR-21 inhibition is being investigated in pre-clinical models of cancer and other diseases associated with miR-21 overexpression [9,10]. A common approach to inhibit miR-21 and other miRNAs is through the use of short single-stranded oligonucleotides (anti-miRs) [11]
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