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. 2019 Mar 13;39(11):2125–2143. doi: 10.1523/JNEUROSCI.1631-18.2018

Figure 6.

Figure 6.

Ago2 mediates circRNA-Filip1l expression by cleavage of the pre-circRNA-Filip1l and regulates the nociceptive behavior. A, Luciferase activities of reporter plasmid after cotransfection of miRNA-1224 mimics, Ago2 overexpression plasmid (Lenti-Ago2) or empty vector (Lenti-vector), and psiCK-mut-pre-Filip1l or psiCK-wt-pre-Filip1l into HEK293T. n = 4 per group. Two-way ANOVA (effect vs plasmid × treated interaction) followed by post hoc Tukey test: *p < 0.05; **p < 0.01. B, Coimmunoprecipitation of AGO2 and pre-circRNA-Filip1l or miRNA-1224. The pre-circRNA-Filip1l, miRNA-1224, and Ago2 complex was pulled down using anti-Ago2 antibody for spinal tissues 3 d after CFA injection. n = 4 per group. **p < 0.01 versus the corresponding Sal groups (two-tailed paired Student's t test). C, D, The expression change of Ago2 protein after intrathecal injection of Lenti-Ago2 in naive mice or CFA-induced pain mice (C), and knockdown of Ago2 with Ago2-siRNA and PLV-Ago2 in naive mice (D). n = 4 per group. *p < 0.05, expression versus the treated groups (one-way ANOVA followed by post hoc Tukey test). Tub, β-Tubulin. E, F, Overexpression of Ago2 decreased the spinal circRNA-Filip1l content (E), but not pre-circRNA-Filip1l content (F) in CFA mice. Spinal cord was collected, respectively, at hour 3 after intrathecal injection of Ago2 protein and day 2 after Lenti-Ago2 intrathecal injection. n = 5 per group. One-way ANOVA (expression vs the treated groups) followed by post hoc Tukey test: *p < 0.05; **p < 0.01. G, H, Knockdown of Ago2 increased the spinal circRNA-Filip1l expression (G), not changed pre-circRNA-Filip1l level (H) day 2 after 2 consecutive days of Ago2-siRNA or PLV-Ago2 injections in naive mice. n = 5 per group. **p < 0.01, versus the corresponding control groups (two-tailed paired Student's t test). I, J, Overexpression of Ago2 alleviated the thermal hyperalgesia and mechanical allodynia induced by intrathecal injection of Ago2 (I) or Lenti-Ago2 for 2 consecutive days (J) in CFA mice. n = 6 per group. Two-way ANOVA (effect vs group × time interaction) followed by post hoc Tukey test: *p < 0.05; **p < 0.01. Red arrow indicates CFA injection. Blue arrows indicate Ago2 or PBS (left) and Lenti-Ago2 or Lenti-vector (right) injections. K, L, Knockdown of Ago2 induced the thermal and mechanical hypersensitivity after 2 consecutive days of intrathecal injections of Ago2-siRNA (K) or PLV-Ago2 (L) in naive mice. n = 6 per group. Two-way ANOVA (effect vs group × time interaction) followed by post hoc Tukey test: *p < 0.05; **p < 0.01. Black arrows indicate Ago2-siRNA or Scr and PLV-Ago2 or vector injections. M, Inhibiting circRNA-Filip1l with anti-Filip1l prevented the thermal and mechanical hypersensitivity induced by knockdown of Ago2 after 2 consecutive days of intrathecal injection of PLV-Ago2 in naive mice. n = 6 per group. Two-way ANOVA (effect vs group × time interaction) followed by post hoc Tukey test: *p < 0.05. Red arrows indicate PLV-Ago2 injection. Blue arrows indicate Anti-Filip1l or Scr injections. N, Overexpression of miRNA-1224 did not change the pain hypersensitivity induced by knockdown of Ago2 with Ago2-siRNA for 2 consecutive days of intrathecal injection in CFA mice. n = 6 per group. Two-way ANOVA (effect vs group × time interaction) followed by post hoc Tukey test. Blue arrows indicate Lenti-1224 or Lenti-vector injections. Red arrows indicate Ago2-siRNA injection.