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. 2015 Jan;43(2):e9.
doi: 10.1093/nar/gku1090. Epub 2014 Nov 6.

Short Loop-Targeting Oligoribonucleotides Antagonize Lin28 and Enable pre-let-7 Processing and Suppression of Cell Growth in let-7-deficient Cancer Cells

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Free PMC article

Short Loop-Targeting Oligoribonucleotides Antagonize Lin28 and Enable pre-let-7 Processing and Suppression of Cell Growth in let-7-deficient Cancer Cells

Martina Roos et al. Nucleic Acids Res. .
Free PMC article

Abstract

MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. Using in vitro assays, we have identified a binding site for short modified oligoribonucleotides ('looptomirs') overlapping that of Lin28 in pre-let-7a-2. These looptomirs selectively antagonize the docking of Lin28, but still permit processing of pre-let-7a-2 by Dicer. Looptomirs restored synthesis of mature let-7 and inhibited growth and clonogenic potential in Lin28 overexpressing hepatocarcinoma cells, thereby demonstrating a promising new means to rescue defective miRNA biogenesis in Lin28-dependent cancers.

Figures

Figure 1.
Figure 1.
Looptomirs targeting the TLR of pre-let-7a-2 inhibit the binding of Lin28A. (a) Predicted structure (Supplementary Figure S9) of pre-miR-122 TLR and binding affinity of 7 10-nt 2′-O-Me-looptomirs measured for binding to biotinylated pre-miR-122 bound to the surface of a streptavidin-coated biosensor by SPR spectroscopy. (SP: position of the looptomir 3′ position). (b) Predicted structure (Supplementary Figure S9) of pre-miR-18a TLR and binding affinity of 8 10-nt 2′-O-Me-looptomirs measured by SPR spectroscopy (SP: position of the looptomir 3′ position). (c) Upper: Predicted structure (Supplementary Figure S9) of the TLR of pre-let-7a-2 showing the sites of interaction of Lin28 zinc finger (Lin28-ZFD)- and cold shock-domains (Lin28-CSD) and Dicer cleavage sites (arrowheads); Lower: looptomirs were assayed for binding to a truncated biotinylated pre-let-7a-2 by SPR spectroscopy (Lx-y refers to a 2′-O-Me looptomir of length y nt, binding with its 3′ terminal nt at position x); binding data of selected looptomirs to pre-let-7a-2 from RNA-based ELISA. (d) SPR sensorgrams from the two strongest binders L29-13 (upper panel) and L30-14 (lower panel) are shown (sensorgrams were analyzed with EVILFIT, as described previously (26)). (e) Principle of the RNA-based ELISA, which measures the IC50 of looptomir oligonucleotides for inhibition of Lin28 binding to immobilized pre-let-7a-2. Full-length Myc-tagged Lin28A from HEK 293 T cell lysate was added to wells and the fraction of bound Lin28A was determined by immunostaining of its Myc-tag. (f) RNA ELISA curves for six selected looptomirs complementary to the TLR depicted in (c).
Figure 2.
Figure 2.
Pre-let-7a-2 is cleaved by Dicer into mature miRNAs in the presence of let-7 looptomirs. (a) Schematic cleavage of pre-miRNAs by Dicer into mature miRNA 5p and 3p strands. (b) In vitro-transcribed pre-let-7a-2 incubated with recombinant Dicer for 150 min: reaction mixtures were analyzed by RP-HPLC, and peaks were identified by MS (inset: HPLC-chromatogram of pre-let-7a-2 used in the study). Pre-let-7a-2 incubated under identical conditions with recombinant Dicer and Lcon (c) and L29-13 (d). Products were analyzed by RP-HPLC. (e) Masses observed and calculated for expected miRNA products from (b).
Figure 3.
Figure 3.
Looptomirs L29-13 and L30-14 induce the expression of functional let-7a. (a) Endogenous levels of mature let-7a 48 h after transfections of L29-13, L30-14 and Lcon in Huh7 and HEK 293T cells, measured by TaqMan RT-qPCR (normalized to RNU44 and Lcon); similar results were found in HepG2 cells (Supplementary Figure S6). (b) Activity of L29-13 and siLin28b in sphere-forming HepG2 cells: TaqMan RT-qPCR of cancer stem-like cells measuring endogenous let-7a levels 4 (left panel) and 6 days (right panel) after transfection of L29-13 at 50 nM concentration. (c) Looptomirs and controls were co-transfected with dual-luciferase vectors into Huh7 cells to measure activity of endogenous let-7. Upper panel: a ‘sensor’ vector containing one let-7a fully complementary target site; lower panel: the same construct mutated at 3 positions in the seed sequence. (d) Western blots showing levels of let-7 target proteins Lin28B and HMGA2 after treatment of Huh7 cells with let-7 looptomirs. (e) Huh7 (left panel) and HepG2 (right panel) cells were transfected with 50 nM of L30-14 and L29-13, as well as pre-let-7a-2 and Lcon controls. OD of stained cells was measured over a 4-day period: OD was normalized to the Lcon treatments. Error bars are SD; ***: P < 0.001. (f) Huh7 (upper panels) and HepG2 cells (lower panels) were transfected with 0.8, 3.1, 12.5 and 50 nM of looptomirs and controls. Colonies were stained with crystal violet and counted after 10 days; cell numbers were normalized to Lcon treatments (toxicity was observed in the 50 nM treatment of Huh-7 cells). Error bars are SD; ***: P < 0.001.

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