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

Figure 4.

Figure 4.

miRNA-1224 is an upstream negative regulator of circRNA-Filip1l. A, Schematic presentation of miRNA-1224 (miR-1224) binding to the fragment of pre-circRNA-Filip1l (pre-ciR-Filip1l). Underline indicates circRNA-Filip1l. Blue represents the reverse complementary of miR-1224 to pre-ciR-Filip1l. B, Distribution of miRNA-1224 in the nucleus and cytoplasma of spinal neuron cultured in vitro. n = 4 per group. Spinal nucleus and cytoplasma RNA were separated from spinal neurons cultured in vitro for 48 h. C, miRNA-1224 FISH in the spinal neuron cultured in vitro. D, CFA induced the time-dependent decrease of spinal miRNA-1224. n = 5 per group. *p < 0.05; **p < 0.01 versus the corresponding Sal groups (two-tailed paired Student's t test). E, Combining FISH of miRNA-1224 and immunofluorescence staining (IF) of NeuN. Scale bar, 25 μm. F, The validation of miR-1224 negatively regulating circRNA-Filip1l by luciferase reporter assay in vitro. A fragment of pre-circRNA-Filip1l containing the bound region by miRNA-1224 was inserted into psiCHECK reporter vectors (psiCK-wt-pre-Filip1l). A mutation was generated via altering the sequence bound by miRNA-1224 as indicated (psiCK-mut-pre-Filip1l). The wild and mutation reporters were cotransfected into the HEK293T with miRNA-1224 mimics or inhibitor or the scrambled. n = 4 per group. Two-way ANOVA (effect vs plasmid × treated interaction) followed by post hoc Tukey test: *p < 0.05. G, Test for the binding capacity of miRNA-1224 to pre-circRNA-Filip1l in vivo. Spinal cord was harvested at hour 24 after intrathecal injection of Bio-miRNA-1224 (Bio-1224) probes, fixed by formaldehyde, and the bound pre-circRNA-Filip1l was pulled down by Dynabeads M-280 streptavidin. n = 4 per group. *p < 0.05 versus Sal group (two-tailed paired Student's t test). H, In vitro transfection of miRNA-1224 mimics or Lenti-1224 enhanced the miRNA-1224 level in HEK293T at hour 48 after treatment. n = 4 per group. **p < 0.01 versus the corresponding groups (two-tailed paired Student's t test). I, Intrathecal injections of miRNA-1224 mimics or Lenti-1224 for 2 consecutive days increased the miRNA-1224 content in spinal cord of naive mice. n = 4 per group. One-way ANOVA (expression vs the treated groups) followed by post hoc Tukey test: *p < 0.05; **p < 0.01. J, K, Intrathecal injections of miRNA-1224 mimics (J) or Lenti-1224 (K) for 2 consecutive days inhibited the expression of spinal circRNA-Filip1l but did not change the spinal pre-circRNA-Filip1l level day 2 after last injection in CFA mice. n = 5 per group. One-way ANOVA (expression vs the treated groups) followed by post hoc Tukey test: *p < 0.05; **p < 0.01. L, M, Intrathecal injections of miRNA-1224 inhibitor (L) or PLV-1224-sponge (M) for 2 consecutive days increased the expression of spinal circRNA-Filip1l but did not change the spinal pre-circRNA-Filip1l level day 3 after last injection in naive mice. n = 5 per group. *p < 0.05 versus the corresponding control groups (two-tailed paired Student's t test).