Neuropathy Pain Associated Micrornas In The Study Of The Features Of The Sensory Nerve Pathway

BackgroundNeuropathic pain affects up to5%of the population and causes substantial disability, but current treatment is inadequate. Chronic neuropathic pain is associated with global changes in gene expression in different areas of the nociceptive pathway. MicroRNAs (miRNAs) are small (-22nt long) non-coding RNAs, which are able to regulate hundreds of different genes post-transcriptionally. The spinal dorsal horn is activated during neuropathic pain and it is an important region for central sensitization. In chronic pain states, painful stimuli trigger afferent fibers in the dorsal horn to release neuropeptides and neurotransmitters. These events induce multiple inflammatory and neuropathic processes in the spinal cord dorsal horn and trigger modification, together with plasticity of local neural circuits. As a result, ongoing noxious signals to the brain are amplified and prolonged, a phenomenon known as central sensitization. The goal of this study was to determine the miRNA expression patterns in the different regions of the pain transmission pathway in the rat bilateral sciatic nerve chronic constriction injury model of rodent neuropathic pain. MiRNA microarray and real-time PCR analyses revealed substantially (-10-fold) decreased miR-203expression in the spinal dorsal horns exclusively and showed that miR-203directly targets the3’-untranslated region of the Rapla gene decreasing Rapla protein expression in nerve growth factor differentiated-neuronal PC12cells. So our study suggested that Rapla gene might be a pain associated gene expressing in spinal horn and could alert the pain threshold under noxius stimuli.Objective:1. The goal of this study was to determine the miRNA expression patterns in the different regions of the pain transmission pathway in the rat bilateral sciatic nerve chronic constriction injury model of human neuropathic pain.2. to figure out miRNA that is substantially changed expression in the spinal dorsal horns, but not the dorsal root ganglions, hippocampi, or anterior cingulate cortexes of bCCI rats, using miRNA microarray and real-time PCR analyse. Then we predict its target gene that is associated with neuropathic pain.3. Investigate this protein expression in spinal dorsal horn of bCCI rats to verify its function during neuropathic pain development. 4. Construct the3’UTR of target gene to a miR-Selection lentivector that contains dual reporter system with firefly luciferase (Fire) and a cytotoxic sensor (Ctx), which enabels the accurate identification of microRNA target.5. To estimate whether the interested miRNA can suppress the protein expression of its target gene of neuronal PC12cell in vitro.Methods:1. To establish the animal modes of neuropathic pain with, the rat bilateral sciatic nerve chronic constriction injury to measure the mechanical hypersensitivity and cold allodynia.2. At the end of the behavioral testing rats were euthanized and the bilateral L4-6dorsal root ganglion (DRG), L4-6spinal dorsal horn, hippocampus and anterior cingulate cortex (ACC) were chronologically harvested rapidly frozen at-180℃for RNA extraction.3. Using microarray and QPCR, we determined the changed miRNAs expression in the different regions of the pain transmission pathway in bCCI rats.4. Finding out the most significantly changed miRNA in the spinal dorsal horn, and then we predict its pain-associated target gene using miRNA databases.5. We estimated the protein expression of target gene in the spinal dorsal horn of bCCI rats. After that we construct the3’UTR of target gene to a miR-Selection lentivector to transfect NGF-differentiated PC12cell.6. Finally, specific miRNA mimics was transfected into neuronal PC12cell to investigate if it can repress target protein expression in vitro.7. Statistical software:SPSS18.0.Results:1. The neuropathic pain model was established successfully, as the threshold of mechanical hypersensitivity and cold allodynia reduced significantly.2. A significant up-regulation of miR-341expression was observed in the dorsal root ganglion, but not in the spinal dorsal horn, hippocampus or anterior cingulated cortex, in neuropathic pain rats compared to naive and sham groups. In contrast, the expression of miR-203, miR-181a-l*and miR-541*was significantly reduced in the spinal dorsal horn of neuropathic pain rats.(fold change>2, P<0.05)).3. MiR-203expression was substantially (-10-fold) decreased in spinal dorsal horn. Rap la protein expression was up-regulated in bCCI rat spinal dorsal horns. According to miRNA database, Rapla was a valid target gene of miR-203.4. We further verified that miR-203directly targets the3’-untranslated region of the Rapla gene decreasing Rapla protein expression in nerve growth factor differentiated-neuronal PC12cells.Conclusions:Every region along with nociceptive pathway has specific and restricted expression of miRNAs associated with neuropathic pain. These data also suggested a potential role for miR-203in regulating neuropathic pain development, and Rapla was a validated target gene in vitro. Results from our study and others indicate the possibility that Rapla/MEK/ERK may be involved in pain and hyperalgesia. We hope that these results can provide support for future research into miR-203in gene therapy for neuropathic pain.