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. 2023 May 5;29(10):2955–2971. doi: 10.1111/cns.14235

FIGURE 6.

FIGURE 6

Upregulated miRNA‐26a was required for CCI‐induced decreases in Kcnk1 expression in nerve‐injured spinal cord. (A) The sequence analysis of miRNA‐26a matching to 3′UTR of nine potassium ion channel mRNA by seven prediction programs including PITA, RNA22, miRmap, microT, miRanda, PicTar, and TargetScan. (B) The analysis of species conservation of miRNA‐26a targeting 3′UTR of Kcnk1 mRNA among four mammal animals. (C) Peripheral nerve injury downregulated the expression of Kcnk1 mRNA in a time‐dependent manner in the ipsilateral dorsal spinal cord after chronic constriction injury (CCI). n = 6 mice/time point/group. *p < 0.05, versus the corresponding Sham group by two‐way ANOVA followed by post hoc Tukey test. (D) The KCNK1 protein decreased in the ipsilateral dorsal spinal cord after CCI. n = 4 mice/group. *p < 0.05 versus the Sham group. (E) Co‐expression analysis of ciRNA‐Kat6b, miRNA‐26a, and Kcnk1 in the single spinal neuron by single cell‐RT‐PCR. The number presented different samples. H2O was used for the negative control. Gapdh was used as the reference gene. (M) DNA ladder marker. (F) Verification of Kcnk1 as a target gene of miRNA‐26a by luciferase reporter assay after the co‐transfection of luciferase reporter (CHK‐wt‐Kcnk1) with miRNA‐26a mimics (miR‐26a mics) or its scrambled control and miRNA‐26a inhibitor (miR‐26a Ih) or its scrambled control in HEK293T cells. A mutation (CHK‐mut‐Kcnk1) was generated in the miRNA‐26a matched seed sequence of Kcnk1 mRNA‐3′UTR. n = 4. *p < 0.05, ***p < 0.001 versus the corresponding group by two‐way ANOVA followed by post hoc Tukey test. (G, H) The expression of Kcnk1 mRNA (G) and protein (H) on day 2 after intrathecal injection of miRNA‐26a inhibitor (miR‐26a Ih) or its scrambled control (Scr) into the mice with 7‐day CCI or Sham surgery mice. n = 6 (G) or n = 5 (H). *p < 0.05, **p < 0.01 versus the corresponding group by one‐way ANOVA followed by post hoc Tukey test. (I, J) The expression of Kcnk1 mRNA (I) and protein (J) on day 2 after intrathecal injection of miRNA‐26a mimics (miR‐26a‐mimics) or its scrambled control (Scr) into naïve mice. n = 6 (I) or n = 4 (J). *p < 0.05, **p < 0.01 versus the scrambled control group by one‐way ANOVA with post hoc Tukey test.