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. Author manuscript; available in PMC: 2015 Apr 2.
Published in final edited form as: Neuron. 2014 Apr 2;82(1):47–54. doi: 10.1016/j.neuron.2014.02.011

Figure 4. Activity-dependent release of let-7b in DRG cultures and involvement of endogenous let-7b in TRPA1 function and inflammatory pain.

Figure 4

(A) Inhibition of formalin (0.01%)-induced inward currents by let-7b inhibitor (anti-mmu-let-7b-5p, 1 μM). Right, amplitude of formalin-induced currents after being normalized to control. *P<0.05, n = 12–15 neurons. (B) Blockade of let-7b- but not miR-599-induced currents in AITC-sensitive DRG neurons by let-7b inhibitor. n = 7–8 neurons. (C) let-7b expression levels in DRG, spinal cord, cortex, and skin tissues, assessed by quantitative RT-PCR and normalized to miRNA SNORD96A (a housekeeping miRNA). *P<0.05, compared to spinal cord, cortex, and skin, n = 3 mice. (D) let-7b release in DRG cultures at different neuronal densities before and after formalin treatment (0.01%, 30 min). let-7b levels in culture medium were assessed by quantitative RT-PCR and normalized to spike-in cel-miRNA-39. *P<0.05, two-way ANOVA, n = 3 cultures. (E) let-7b and let-7a release (fold of control) in DRG culture medium following 30 min treatment of formalin (0.01%), ionomycin (10 ng/ml), KCl (50 mM), capsaicin (1 μM), AITC (300 μM), or chloroquine (1 mM). *P<0.05, compared to control, n = 4–12 cultures. (F) let-7b-induced spontaneous pain at different doses. *P<0.05, vs. vehicle; #P<0.05, n.s., not significant. (G and H) Reduction of let-7b (30 μg)-induced spontaneous pain in Trpa1−/−, Tlr7−/−, and Myd88−/− mice but not in Trpv1−/− mice. *P < 0.05, vs. WT control; #P<0.05. (I) Dose-dependent inhibition of let-7b-evoked spontaneous pain by intraplantar HC030031 (1, 10 μg). *P<0.05, vs. let-7b vehicle (RNase free-water); #P<0.05, vs. HC030031 vehicle (5% DMSO, green bar). (J) Low-dose let-7b (1 μg) induced mechanical allodynia in WT mice and Trpa1−/− mice. *P<0.05; #P<0.05. (K and L) High-dose let-7b (30 μg) induced mechanical allodynia in WT mice and Tlr7−/− (K) and Trpa1−/− (L) mice. *P<0.05; #P<0.05. (M) Inhibition of formalin (0.5%) induced pain in Phase I (0–10 min) and Phase II (10–45 min) by intraplantar let-7b inhibitor (3 nmol). *P<0.05. (N) Formalin (0.5%) induced pain in Phase I (0–10 min) and Phase II (10–45 min) in WT and Tlr7−/− mice. *P<0.05. The data are mean ± SEM. n = 5–11 mice for behavioral studies in panels F–N.