MiR-137 Impacts Neuropathic Pain By Regulating Voltage-gated Potassium Channel Kv1.2 In Rats

BackgroundNeuropathic pain is directly caused by the injuries or diseases of the somatosensory system and causes clinical features including spontaneous pain,hyperalgesia,allodynia and paresthesia etc,resulting in badly diminished quality of patients'life.However,due to the unclarity in the mechanism,medication for neuropathic pain still gets unideal effects.Therefore,deep investigations are in desperate need to provide new ideas and methods for effective manegement.The abnormity of multiple ion channels play key roles in the initiation and maintanance of neuropathic pain,among which the voltage-gated potassium channels occupy an important position.Thereinto,Kv1.2 attracts widely attention for its role in maintaining resting membrane potential,mediating rapid repolarization of action potentials and modulating neuronal excitability.Our preliminary experiments showed significant reductions in the expression of Kv1.2 in chronic constriction injury?CCI?rats.Neverthless,how the Kv1.2 is regulated in neuropathic pain still waiting to be explored.MicroRNAs?miRNAs?regulate the expression of genes by completely or incompletely pairing with the 3?UTR sequence of mRNAs.In recent years,a large number of literatures have reported that a variety of miRNAs are involved in neuropathic pain,leading to a new direction for pain treatment.Using the bioinformatics software?Target Scan Human 7.1?,the 3?UTR sequence of KCNA2?Kv1.2 gene?mRNA and miR-137 were found to be highly correlated,suggesting that KCNA2 is a potential target for miR-137.Therefore,we hypothesize that miR-137,through regulating the expression of KCNA2,changed the level of Kv1.2 protein and the neuronal Kv current,thus participating in neuropathic pain.PurposeIn this study,we evaluate wether miR-137 regulates the expression of Kv1.2 and how they participate in the CCI-induced neuropathic pain,thus to provide new targets and theoretical basis for the treatment of neuropathic pain.Methods?1?Rat CCI neuropathic pain model was established.The changes of mechanical and thermal pain threshold at different time points after surgery were detected.?2?Whole-cell voltage-clamp recording was used to test the total Kv current and TEA-sensitive Kv current in the L4-L6 DRG neurons in sham and CCI rats.?3?Using q-PCR and western blot,we compared the difference of Kv1.2 mRNA and protein between ipsilateral and contralateral spinal cord and DRGs?CCI 3d?,and detected the change of Kv1.2 mRNA and protein in the ipsilateral spinal cord and DRGs after CCI at different time points?CCI 3d,7d and 14d?.?4?We gave KCNA2 siRNA to normal rats and detected the pain behavior performance by behavioral test and the Kv1.2 protein change by western-blot.?5?We checked the distribution of Kv1.2 in DRG before and after KCNA2siRNA injection using immunofluorescence.?6?q-PCR was used to test the expression level of miR-137 at the 3d,7d and 14d after CCI surgery.?7?We used the dual-luciferase reporter gene to check whether miR-137has a target relationship with Kv1.2.?8?To evaluate whether miR-137 has a regulatory role on Kv1.2,normal or TNF-treated primary DRG neurons were cultured and given the miR-137 agomir or antagomir respectively followed by the Kv1.2 protein level tests using western blot.?9?MiR-137 agomir was injected into normal rats before we tested the changes of mechanical and thermal pain threshold by behavioral test,the Kv1.2 protein expression level in spinal cord and relative DRGs by western-blot,and the Kv current in relative DRGs by whole-cell voltage-clamp recording.?10?MiR-137 antiagomir was given to CCI rats followed by the behavioral tests of mechanical and thermal pain threshold,the western-blot analysis for the Kv1.2protein expression level and the whole-cell recording of Kv current.Results?1?When compared with the basic pain threshold,both mechanical and thermal pain threshold of the ipsilateral posterior claw started to show significant reduces since the 3^rdday after CCI surgery,reached the minimum at the 7^thday and lasted until the 14^thh day?P<0.001?.However,on the contralateral side,neither mechanical pain threshold nor thermal pain threshold were changed?P>0.05?.In the Sham group,the pain threshold kept stable at normal level during the detection,proving that our CCI model was successful.?2?When compared with sham group,the total and TEA-sensitive Kv current in L4-L6 DRGs of CCI rats were significantly decreased.?3?Both the mRNA and protein levels of Kv1.2 in the ipsilateral spinal cord and relative DRGs at CCI 3d were significantly lower than that in the contralateral side.The Kv1.2 mRNA and protein in the ipsilateral spinal cord and relative DRGs significantly reduced at CCI 3d,7d,and 14d when compared to the sham group.?4?Intrathecal injection of KCNA2 siRNA induced a significant decrease in the mechanical and thermal pain threshold of bilateral posterior claws on normal rats.By western–blot,we also detected a decrease in Kv1.2 protein level in the spinal cord and DRG?P<0.001?.?5?Immunofluorescence results showed that,in DRG tissues,Kv1.2 was mostly expressed in large and medium-sized neurons while a little was expressed in peptide neurons and no Kv1.2 was co-labeled with the small neuron marker IB4.?6?When compared with the sham group,the expression level of mir-137 in the spinal cord and DRG tissues at CCI 3d,7d and 14d were significantly increased.?7?Dual-luciferase reporter?DLRTM?showed that the luciferase activity of PC12 cells transfected with wild-type KCNA2 carrier and miR-137agomir significantly decreased?P<0.01?,while the luciferase activity of cells transfected with mutant KCNA2 carrier and miR-137agomir did not change?P>0.05?,showing that miR-137 can target the KCNA2 3'UTR directly.?8?Kv1.2 protein level were decreased by miR-137agomir treatment?P=0.0042?while the N.C group showed no difference.?9?The TNF-?-induced sharp fall of Kv1.2 protein in primary DRG neurons?P<0.01?while it can be reversed by miR-137antagomir treatment?P=0.0035?,verifying the regulating role of mi R-137 on Kv1.2.?10?In vivo experiment results showed that miR-137agomir injection caused severe mechanical and thermal hyperalgesia in normal rats,accompanied with significant reduction of the mechanical and thermal pain threshold,the Kv1.2 protein?P<0.05?both in spinal cord and DRGs and the Kv current in relative DRG neurons.There's no difference found in the Na?ve+N.C group.?11?Injection of miR-137antagomir significantly reversed the CCI-induced mechanical and thermal hyperalgesia,accompanied with remarkable rise in the mechanical and thermal pain threshold,the Kv1.2 protein?P<0.05?both in spinal cord and DRGs and the Kv current in relative DRG neurons.These results verified again that miR-137 could regulate Kv1.2 and participate in the CCI-induced neuropathic pain.ConclusionIt can be concluded that both Kv1.2 and mi R-137 are involved in neuropathic pain,and mi R-137 can regulate Kv1.2,suggesting us a novel way to regulate Kv1.2expression by up-or down-regulating mi R-137 and manage neuropathic pain.