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. 2016 Mar 2;36(9):2769–2781. doi: 10.1523/JNEUROSCI.3474-15.2016

Figure 4.

Figure 4.

DNA hydroxymethylation mediated by TET1 and TET3 in the miR-365-3p promoter regulates spinal miR-365-3p expression and acute pain behavior. A, The activities of the methylated or demethylated promoter of the cloned miR-365-3p promoter encompassing TSS of pre-miR-365-3p were detected by firefly luciferase reporter assays in HEK293T cells. The pGL6 plasmid (empty vector) was used as the negative control; pGL6-365, plasmid with miR-365-3p promoter. 5C, Unmethylated group; 5mC, methylated group; 5hmC, hydroxyl-methylated group. Values of luciferase activities for each plasmid were normalized for transfection efficiency by cotransfection with pRL-TK plasmid. **p < 0.01 versus adjacent groups, ***p < 0.001 versus negative group; n = 6. B, Tet1- and Tet3-siRNA reversed the increase in spinal 5hmC of miR-365-3p caused by formalin injection. The relative iv5hmC level was measured using hMeDIP-qPCR. Input represents PCR products from genomic DNA without hMeDIP. **p < 0.01 versus control group, #p < 0.05 versus FM+Scr; n = 6. C, TET1 and TET3 overexpression mediated by lentivirus increased the 5hmC level of the miR-365-3p promoter in naive mice. *p < 0.05 versus the Lenti-vector group; n = 6. D, Downregulation of spinal TET1 and TET3 induced by siRNA decreased the expression of miR-365-3p. *p < 0.05 versus the FM+ Tet1-siRNA or Tet3-siRNA group; n = 6. E, Upregulation of spinal TET1 and TET3 induced by lentivirus increased the expression of miR-365-3p. **p < 0.01 versus the Lenti-vector group; n = 6. F, Time of climbing test at 72 h after intrathecal (i.t.) injection of miR-365-3p inhibitor (Ih), miR-365-3p mimics (mics) or Scr for 3 consecutive days, or at 5 min after intraplantar (i.pl.) injection of miR-365-3p mimics or Scr in naive mice. No significance versus Ctrl or Scr group; n = 6. G, Intrathecal injection of miR-365-3p inhibitor for 3 consecutive days alleviated formalin-induced acute pain in phase 2. *p < 0.05 versus FM+ Scr group, **p < 0.01 versus FM+Scr group; n = 6. H, Intrathecal injection of miR-365-3p mimics for 3 consecutive days induced the production of thermal hyperalgesia in naive mice. Black arrow indicates miR-365-3p mimics or Scr injection. *p < 0.05 versus Scr group, **p < 0.01 versus Scr group; n = 8. I, Intraplantar injection of miR-365-3p mimics produced pain-like behavior. Five microliters of miR-365-3p mimics or Scr (20 μm) was injected. **p < 0.01 versus Scr group; n = 8. J, Intrathecal injection of miR-365-3p inhibitor markedly inhibited thermal hyperalgesia induced by Lenti-T1 and Lenti-T3 in naive mice. Black arrow indicates Lenti-T1 and Lenti-T3 injection; blue arrow indicates miR-365-3p inhibitor or Scr injection. *p < 0.05 versus Lenti-T1 or Lenti-T3+Scr, **p < 0.01, versus Lenti-T1+Scr; n = 8.