SiRNA-mediated ankyrin-G silence modulates the expression of voltage-gated Na channels in murine hippocampal HT22 cells

Abstract

Background: Voltage-gated sodium channels are the targets of many commonly used antiepileptic drugs. NaV1.6, encoded by Scn8a, increased in chronic mesial temporal epilepsy animal models and co-localized with Ankyrin-G, encoded by Ank3. We hypothesized that inhibition of Ank3 transcription by siRNA decrease the expression of NaV1.6. Results: We characterized expression of the target genes in hippocampal neuron HT22 cells by Real time-PCR. The melt peak in the resolution curve of Scn1a, Scn8a and Ank3 were all unique. Ank3 transcription was interfered and the relative Ank3 mRNA levels of the three interfered groups compared to GAPDH were 0.89 ± 0.13, 0.52 ± 0.07 and 0.26 ± 0.05 while that of the negative control group was 1.01 ± 0.08 (P < 0.05). When Ank3 transcription was inhibited by siRNA, the relative mRNA levels of Scn8a decreased in the three groups (0.91 ± 0.09, 0.33 ± 0.06 and 0.25 ± 0.05), compared to the negative control group (1.10 ± 0.09). Tested by Western blotting, protein levels of ankyrin-G and Nav1.6 decreased after ank3-siRNA. Ankyrin-G in negative control group, group1, group2 and group1 + 2 were 0.813 ± 0.051, 0.744 ± 0.041, 0.477 ± 0.055 and 0.351 ± 0.190 respectively (P < 0.01) while Nav1.6 were 0.934 ± 0.036, 0.867 ± 0.078, 0.498 ± 0.070 and 0.586 ± 0.180 (P < 0.01). The quantity analysis of immunofluorescence showed significant decrease of ankyrin-G and Nav1.6 (Student's test, P = 0.046 and 0.016 respectively). Conclusion: We therefore concluded that in HT22 cells the expression of Nav1.6 was down-regulated by Ank3 RNA interference.