Article Figures & Data
Figures
- Figure 1.
Interaction of let-7 with the 3′-UTR of the MOR. A, Sequence alignment of the let-7 family miRNAs and the predicted sequence pairing with a region of MOR mRNA 3′-UTR. The nucleotides in red are the positions that are identical among all members of the let-7 family. The predicted MOR mRNA 3′-UTR sequences that may interact with let-7 seed match are in green (bold). B, A schematic plot of the luciferase constructs. Solid bar indicates the location of the putative let-7 binding site within the 3′-UTR of MOR mRNA. The LNA-let-7 inhibitor and scrambled-LNA control are shown below the let-7a alignment with the position of LNA nucleotides underlined in bold. C, Luciferase reporter assays were performed in HEK293 cells transfected with the reporter plasmids psiCHECK/MOR1UTR or psiCHECK/MOR1UTRmut together with the LNA-let-7 inhibitor (0, 1, 5, 25 pmol) or scrambled-LNA control (25 pmol). The Renilla luciferase activity was normalized to the internal control firefly luciferase activity. *p < 0.05, ***p < 0.001 vs the 3′-UTR control (n = 3).
- Figure 2.
Constitutive repression of MOR expression by let-7 miRNAs in SH-SY5Y cells. SH-SY5Y cells were transfected with the LNA-let-7 inhibitor (0, 2, 10, 50, 250 pmol) or scrambled-LNA control (250 pmol). Cells were lysed for Western blot analysis 48 h later. The anti-MOR antibody did not detect 65 kDa MOR bands in the null deletion mice (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Histogram graph was constructed from the representative figure shown and two other experiments. ***p < 0.001 vs the control.
- Figure 3.
Increased let-7 expression and decreased MOR expression in SH-SY5Y cells during the development of opioid tolerance. SH-SY5Y cells with or without retinoic acid (RA) pretreatment (5 μm, 6 d) were incubated with morphine (1 μm, 24–48 h) to induce a tolerant state. The transcripts of let-7a (A), let-7c (B), and let-7g (C), but not miR-134 (D), were increased as determined by the real time RT-PCR. MOR expression was decreased as measured by Western blot analysis (E, F). *p < 0.05, **p < 0.01, ***p < 0.001 compared with 0 h without RA; ##p < 0.01, ###p < 0.001 compared with 0 h with RA (n = 3).
- Figure 4.
Increased let-7 expression in the mouse brain during the development of opioid tolerance. Mice were implanted with morphine pellets to induce opioid tolerance. On day 0, 1, 3 and 5, brain let-7a (A), let-7c (B), let-7g (C), and miR-134 (D) transcripts were analyzed by quantitative RT-PCR. The development of opioid tolerance was confirmed by following significantly reduced morphine-antinociception in a tail-flick assay (E). *p < 0.05, ***p < 0.001 vs day 0 group (n = 5).
- Figure 5.
Fluorescence in situ hybridization (FISH) of let-7 before and after the treatment with morphine (75 mg/pellet/mouse). The brain cortex was used for the analysis of let-7 mRNA by FISH. Samples were costained using anti-MOR antibody (1:5000) to determine the level of let-7 in MOR-expressing cells. The let-7 transcript was increased after morphine treatment. Moreover, the increase was observed in MOR-expressing cells (arrows indicate representative cells with costaining of MOR and let-7), but not in MOR-negative cells (only DAPI nuclear staining, but no MOR staining). Fifteen prefrontal cortex slices from 3 mice were studied for each condition. In the control group, 78 MOR-expressing cells and 60 MOR-negative cells were imaged. In the morphine group, let-7 upregulation occurred in 78/85 MOR-expressing cells; no cell showed let-7 response in MOR-negative cells (0/65).
- Figure 6.
Effect of the LNA-let-7 inhibitor on morphine antinociception, morphine antinociceptive tolerance, and let-7 expression in mice. A, The LNA-let-7 inhibitor potentiated morphine antinociception in naive mice, suggesting constitutive repression of MOR by let-7. B, The LNA-let-7 inhibitor dose-dependently attenuated morphine antinociceptive tolerance. C–E, Morphine-induced let-7 upregulation was dose-dependently reduced by the LNA-let-7 inhibitor (0.3–3.0 nmol, twice/day, for 3 d). *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the morphine group (n = 4).
- Figure 7.
The total and polysome-associated μ opioid receptor transcript after the treatment with morphine in SH-SY5Y cells. SH-SY5Y cells were treated with morphine (1 μm, 48 h). A, MOR transcript was not altered by morphine (n = 5 each group). B, Polysome-associated MOR mRNA was decreased by morphine. The change was significantly attenuated by the LNA-let-7 inhibitor. **p < 0.01 vs the control group; #p < 0.05, vs the morphine group (n = 5 each group). C, Colocalization of MOR mRNA with hDcp1a, a P-body marker, after, but not before, morphine treatment. Blue (DAPI) identifies nuclei.
- Figure 8.
A schematic plot for the proposed mechanism of MOR regulation by let-7. Pri- and pre-let-7 were produced and exported into the cytosol where the mature let-7 was incorporated into the RISC. The latter recruits and sequesters MOR mRNA to P-bodies that are deprived of translational machinery, effectively reducing polysome-bound MOR mRNA and leading to translation repression.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Material
