MicroRNA30b Regulate The Expression Of SCN9A In Neuropathic Pain

Background and purposeThe damage or dysfunction of the central or peripheral nervous system can cause neuropathic pain which is featured by spontaneous pain,hyperalgesia and hypersensitive pain.Its mechanism is so complicated that difficult to be treat. It has been found that single nucleotide mutation of SCN9A is related to the congenital disorders pain. Missense mutations in the SCN9A causes primary erythermalgia (Primary erythermalgia, PE) and paroxysmal extreme pain disorder (paroxysmal extreme pain disorder PEPD), while single gene nucleotide deletion of SCN9A leads to congenital insensitivity (congenital inability to experience pain CIP). Therefore, SCN9A may be a key factor to the occurrence and development of pain. SCN9A gene is located on second chromosome. It is a6000bp length gene with26exons. The alpha subunit of voltage-gated sodium channel Navl.7which encodes tetrodotoxin (TTX)sensitive, is expressed mainly in the peripheral dorsal root ganglia and sympathetic ganglion neurons.MicroRNA(miRNA) is a class of non-coding single stranded RNA molecules encoded by a class of endogenous gene in length of about22nucleotides, mainly involved in regulating gene expression post-transcriptionally. When the miRNA bind to the target mRNA completely, target mRNA will be degraded directly. However, while the miRNA bind to the targer mRNA incomplete, it will prevent protein translation.. We forecast by the biological information software Targetscan, miR-30b and SCN9A had the strongest correlation, therefore we hypothesis that:in the normal physiological condition, miR-30b could inhibit the SCN9A from mRNA translate to Navl.7, maintaining Navl.7homeostasis. After the peripheral nerve injury, the expression of miR-30b reduces the inhibition of SCN9A mRNA, thus increasing the expression of Nav1.7, leading to enhanced to neuronal excitability, resulting in pathological pain.Therefore, in this study, we performed the sciatic nerve injury(SNI) of sciatic nerve on rats,and detect the change of SCN9A mRNA and protein level by over-expressing and inhibiting the expression of miR-30b. Meanwhile,we detected the change of pain threshold in rats to confirm the proposed hypotheses, thus, providing the theoretical foundation for the pathogenesis of neuropathic pain, as well as a new target for the therapeutics. Method(1) For SNI:tibial and peroneal nerve were double ligated. respectively and cut at the central of the double ligation,then removed the end of each neural stem of24mm.(2) Determination of50%paw withdrawal threshold (von Frey test):rats showing rapid flinching reaction in stimulation time were marked as positive. Ac cording to the method reported by Dixon (1980),we calculated the threshold of50%paw withdrawal. When50%paw withdrawal threshold is less than4g,it would be considered as a hyperalgesia.(3) qRT-PCR Currently, qRT-PCR is the most common and important method to detect miRNA,which is fast,specificity and sensitivity.(4) Immunohistochemical double staining:it is used for locating and semi-quantitative detecting Nav1.7.The result can be showed intuitively and validly.(5) Western blot is used for detecting the change of the expression of Navl.7.(6) Dual luciferase reporter gene assay:the luciferin and luciferase bio luminescence system is used to detect the expression of gene sensitively and efficiently. It is a detection method for detecting the interactions between a transcription factors and the DNAin the promoter region of target gene.(7) Intrathecal catheterization:Works on the5th intrathecal spinal syringe needle to drill a piercing,then clip the peak of polyethylene catheter and placed upward gently,then we can see the pure cerebrospinal outflowing within the catheter.Appearance of flick or shake reflection means a successful puncture.The insertion depth of catheter is about1.5-2.0cm.Result1.In neuropathic pain, miR-30b and SCN9A has negative correlation.2.miR-30b has a certain effect on expression of SCN9A3.Intrathecal injection of miR-30b can alleviate neuropathic pain.ConclusionIn vivo and in vitro experiments confirmed that in the course of neuropathic pain, miR-30b involved in regulating the expression of SCN9A,and inhibited SCN9A translated into protein Nav1.7, thus relieving neuropathic pain.