Knockdown of rno_circRNA_009194 Improves Outcomes in Traumatic Brain Injury Rats through Inhibiting Voltage-Gated Sodium Channel Nav1.3

Abstract

Excessive activation of voltage-gated sodium channel Nav1.3 has been recently reported in secondary traumatic brain injury (TBI). However, the molecular mechanisms underlying regulating voltage-gated sodium channel (Nav1.3) have not been well understood. The present study used a TBI rat model induced by a fluid percussion device and performed a circular RNA (circRNA) microarray ( n = 3) to profile the altered circRNAs in the hippocampus after TBI. After polymerase chain reaction (PCR) validation, certain circRNAs were selected to investigate the function and mechanism in regulating Nav1.3 in the TBI rat model by intracerebroventricular injection with lentivirus. The neurological outcome was evaluated by Morris water maze test, modified Neurological Severity Score (mNSS), brain water content measurement, and hematoxylin and eosin staining. The related molecular mechanisms were explored with PCR, Western blotting, luciferase reporter, chromatin immunoprecipitation assay, and electrophoretic mobility shift assay (EMSA). A total of 347 circRNAs were observed to be differentially expressed (fold change [FC] ≥ 1.2 and p < 0.05) after TBI, including 234 up-regulated and 113 down-regulated circRNAs. Among 10 validated circRNAs, we selected circRNA_009194 with the maximized up-regulated fold change ( n = 5, FC = 4.45, p < 0.001) for the in vivo functional experiments. Down-regulation of circRNA_009194 resulted in a 27.5% reduced mNSS in rat brain ( n = 6, p < 0.01) after TBI and regulated the expression levels of miR-145-3p, Sp1, and Nav1.3, which was reversed by sh-miR-145-3p or Sp1/Nav1.3 overexpression ( n = 5, p < 0.05). Mechanistically, circRNA_009194 might act as a sponge for miR-145-3p to regulate Sp1-mediated Nav1.3. This study demonstrated that circRNA_009194 knockdown could improve neurological outcomes in TBI in vivo by inhibiting Nav1.3, directly or indirectly.