Early Over-Expressing of microRNA-145 Effectively Precludes the Development of Neuropathic Mechanical Hyperalgesia via Suppressing Nav1.8 in Diabetic Rats

Abstract

Background Painful diabetic neuropathy (PDN) is a common complication secondary to diabetes mellitus. Nav1.8 is an isoform of voltage-gated sodium channels and its expression regulation is closely related with PDN. MicroRNA-145 (miR-145) is involved in the occurrence and development of neuropathic pain. TargetScan software has revealed that Nav1.8 (SCN10A) is the major target of miR-145. However, its function between miR-145 and Nav1.8 in PDN is unknown. Objectives We aim to explore the regulatory effect of miR-145 on the expression and function of Nav1.8, which plays a pivotal role in precluding the advancement of neuropathic mechanical hyperalgesia in diabetic pain. Study design An experimental, animal study. Setting An animal research facility at Nanjing Maternal and Child Health Institute, China. Methods The paw mechanical withdrawal threshold (PMWT) of rats was assessed with the von Frey test. The adverse regulation of Nav1.8 by miR-145 was confirmed by a dual luciferase detection system in HEK293T cells. The mRNA level and expression of Nav1.8 in dorsal root ganglion (DRG) neurons were assessed with real-time polymerase chain reaction (real-time PCR), western blotting and immunofluorescence assays following intrathecal injection of agomiR-145 in vitro and in vivo. Whole-cell patch-clamping was applied to assess alterations in the tetrodotoxin-resistant (TTX-R) sodium current (Nav1.8) in DRGs. Results The PMWT was significantly decreased in rats following streptozotocin (STZ) injection on Day 7 and was maintained at a lower level on Day 28; this change was accompanied by changes in the expression of Nav1.8 in DRG neurons, which was increased 3 days after STZ injection and reached a maximal level on Day 14. The early knockdown of Nav1.8 with siRNA or agomiR-145 treatment on Day 8 effectively precluded the deterioration of pain behaviors in STZ-treated rats. The luciferase intensity was significantly decreased in HEK293T cells expressing wild-type SCN10A infected with miR-145 mimic. In addition, Nav1.8 overexpression was significantly repressed via overexpression of miR-145 in cultured DRG neurons, and neuronal hyperexcitability was concomitantly decreased. Furthermore, the intrathecal administration of agomiR-145 elicited a significant decrease in Nav1.8 expression in DRG neurons from STZ-treated rats on Day 14. Limitations The causes of PDN are likely to be multifactorial and inflammatory markers, such as IL-6, IL-2, and TNF-?, are elevated in hyperglycemia and might be the precipitating factors that contribute to miR-145 dysregulation. The curative effect of miR-145 upregulation in reversal of pain behaviors at the stage of well-established PDN wasn't investigated in this study. Conclusion Early infection with a lentiviral vector overexpressing miR-145 adversely regulated the expression and function of TTX-resistant Nav1.8 and abrogated the development of PDN. Therefore, miR-145 might be a potential therapeutic target for preventing PDN in the near future.