| Background:The IASP(International Association for the Study of Pain)defines pain as an unpleasant sensory and emotional experience associated with actual or potential tissue damage,or described in terms of such damage[1].Neuropathy pain is a common clinical chronic recalcitrant,persistent pain syndrome.The approximate prevalence of neuropathic pain in the gross population is 7–10%[2,3],remaining extremely difficult to treat,due to barely understood pathogenesis and a lack of well-defined molecular targets.The voltage-gated sodium channels(VGSCs,Nav1.1–Nav1.9 and Nax)containing tetrodotoxin-sensitive(TTX-S)channels and tetrodotoxin-resistant channels(TTX-R)are involved in the generation and propagation of action-potential[4].Nav1.3 is a subunit among the VGSCs,encoded by the SCN3 A gene and located on chromosome 2[5].SCN3 A has a high expression in the central nervous system of embryos and newborns but is poorly expressed in adult rats[5].And it has been reported that neuropathic pain was perhaps caused by the aberrant expression of SCN3A[6].Nav1.3 is re-expressed in DRG neurons after peripheralnerve injury[7,8].Similarly,the level of Nav1.3 increases in spinal dorsal horn neurons following SCI surgery[9,10].In a previous study,the repression of Nav1.3 using Nav1.3-specific antisense(AS)oligodeoxynucleotide(ODN)blocked mechanical and thermal alloydnia[11].Nav1.3 was probably a key molecule involved in neuropathic pain.However,the mechanism of altered Nav1.3 expression continues to perplex.Non-coding RNA(NcRNA)regulating the expression of proteins has emerged as a target.MicroRNAs(miRNAs)are endogenous ncRNAs.They are responsible for the regulation of gene expression through the targeting of the gene 3'UTR[12,13].MiRNAs are highly deregulated in diseases and might be a critical molecule for treatment[14].And recent studies that associated mi RNAs with chronic neuropathic pain attracted wide attention,which provided a new insight for the treatment of neuropathic pain.Using Target Scan software,we found that miR-30 b was closely related to SCN3 A.As a result,we propose such a hypothesis: under normal physiological condition,miR-30 b inhibits the translation of SCN3 A mRNA to maintain a steady state for Nav1.3 expression;after peripheral nerve injury,miR-30 b was downregulated with the increase of Nav1.3 mRNA and protein as well as the enhanced excitability of neurons,leading to neuropathic pain.Research purposesIn the present study,we intended to creat an SNL(spinal nerve ligation)model and clarify the changes of mi R-30 b and Nav1.3 in neuropathic pain induced by SNL;experiments in vitro were used to determine the relationship between miR-30 b and SCN3A;then we meaned to evaluate changes in Nav1.3 mRNA and protein expression by overexpressing or inhibiting miR-30 b,and understand the sensitivity of rats to mechanical and thermal stimuli,then validated our hypothesis.On the other hand,the role of endogenous miR-30 b in regulating the expression of SCN3 A was identified to explore the specific mechanism in the development and maintance of neuropathic pain at the molecule level,providing a new insight for improving therapeutic approaches to pain.Research methods(1)Bioinformatics software was used to determine the miRNAs related to SCN3 A.Using Target Scan software(Taget Scan Human 7.1),miRNAs associated with SCN3 A were predicted.(2)The creation and identification of SNL model in rats.Animals were devided into two groups randomly,sham/SNL.As for SNL rats,after the animals were anesthetized,the transverse process of the left L6 was removed to expose the L4 and L5 spinal nerves.After isolating the left L5 spinal nerve,a tight ligature was made with 3-0 silk,and the nerve was transected distal to this ligature.In the sham-operated group,the left L5 spinal nerve was separated but remained complete and unscathed with no ligature or transection.We assess whether the model was conducted successfully by determining the behaviors including 50% mechanical paw withdrawal threshold(PWTs)and thermal paw withdrawal latency(PWLs);qRT-PCR and western-blot were used to measure the expression changes of Nav1.3 at mNA and protein level both in dorsal root ligation(DRG)neurons and spinal cord;moreover,immunofluorescence and in situ hybridization were performed to define the localization of Nav1.3 and mi R-30 b in DRG neurons and spinal cord.(3)Luciferase assay was applied to assess whether miR-30 b could target SCN3 A.Using the pmirGLO dual-luciferase vector containing the sequence of SCN3A3'UTR.Co-transfection of miRNA mimics(miR-30 b agomir)at different doses of 10 pM,50 pM,and 100 pM(50 nmol/L)with wild-type reporter vectors(0.5 ?g/mL)was performed with Invitrogen lipofectamine 2000 in PC12 cells.Then co-transfection of other miRNAs with wild-type and mutant-type reporter vectors was conducted without serum medium or antibody as per the manufacturer's instructions.The ratio of firefly activity to renilla activity was recognized as relative reporter activity.(4)Culture and transfection of primary DRG neurons were performed to explore the relationship between miR-30 b and SCN3 A in vitro.To determine whether miR-30 b may regulate the expression of Nav1.3,on one hand,TNF-?(2 ?L,100 ng/mL)was used to stimulate the primary DRG neurons;30min later,we transfected mi R-30 b agomir,qRT-PCR and western-blot were used to determine the changes in the expression of miR-30 b,Nav1.3 m RNA and protein after the stimulation of TNF-? to assess the impact of miR-30 b agomir on the expression of Nav1.3 with TNF-? stimulation.On the other hand,we transfected miR-30 b antagomir into normal DRG neurons,and measured the expression of Nav1.3 mRNA and protein.(5)Experiments in SNL rats were performed to explore the exact impact of miR-30 b on neuropathic pain induced by SNL.MiR-30 b agomir(20 ?M,10 ?l)was delivered to SNL rats for 4 days following day 10 with intrathecal injection,and 50% PWTs and PWLs were tested.We evaluated the analgesic effect of miR-30 b through the determination of changes in behaviors and molecules,qRT-PCR and western-blot were performed to assess the impact of mi R-30 b agomir on the expression of miR-30 b and Nav1.3 both in DRG neurons and spinal cord in SNL rats,exploring the underlying mechanism of miR-30 b interacted SCN3 A in neuropathic pain;inversely,we down regulated miR-30 b by intrathecal injection with mi R-30 b antagomir in na?ve rats.MiR-30 b antagomir was applied to na?ve rats for 4 days and determined their sensitivity to mechanical and thermal stimulus,as well as the changes in the expression of Nav1.3 by qRT-PCR and western-blot.Results(1)It was predicted that SCN3 A was highly related to mi R-30abcde-5p/384-5p,miR-96/182/183-5p and miR-132-3p/223-3p using Target Scan Human 7.1.(2)SNL model was created in success with the increased expression of Nav1.3and the decreased expression of miR-30 b.Compared with sham-operated rats,SNL induced a conspicuous reduction in50% PWTs and PWLs of the ipsilateral hindpaw of the injured side(***P<0.0001)(from 3 d to 21d),indicating that SNL model was conducted successfully,simultaneously,qRT-PCR and western-blot analysis showed that SNL caused an obvious downregulation of miR-30 b expression and upregulation of Nav1.3 mRNA and protein expression in DRG neurons,as well as in the spinal cord;we stainedNav1.3 with NF-200(a marker of large myelinated non-nociceptive neurons),IB4(a marker for non-myelinated nociceptive neurons),CGRP(a marker for nociceptive Peptide neurons)and GS(a marker for glial cells)respectively.Double-labeled immunofluorescence showed that Nav1.3 signal was mainly double-labeled with IB4 and CGRP,which are markers for non-myelinated nociceptive neurons,while it was not found to localize with NF-200 and GS,moreover,in situ hybridization results expressed that miR-30 b was double-labeled with NF200,IB4,and CGRP.Importantly,the cells containing miR-30 b express Nav1.3 in DRG neurons.(3)Luciferase assay showed that miR-30 b could target SCN3 A.Transfecting the wild-type plasmid vector with three different doses of miR-30 b agomir(10 pM,50 pM,100 pM)into PC12 cells with Lipofectamine 2000,miR-30 b agomir reduced relative luciferase activity in a dose-dependent manner(***P<0.0001).However,the luciferase activities of miR-30 b antagomir and scrambled miRNAs were unchanged(P>0.05)indicating that the inhibition of miR-30 b agomir was sequence specific.To further prove the specificity of SCN3A3'UTR,we transfected mutant 3'UTR plasmid with miR-30 b agomir into PC12 cells.As expected,miR-30 b had no effect on luciferase activity.(4)MiR-30 b agomir inhibited the expression of Nav1.3 in primary DRG neurons.Compared to the na?ve non-transfected group,TNF-? stimulation induced a significant increase in Nav1.3 at mRNA(***P=0.0003)and protein level(***P<0.0001)while a reduction of miR-30 b was observed(***P=0.0007)in primary DRG neurons.However,miR-30 b overexpression,by transfecting mi R-30 b agomir,reversed the upregulation of SCN3A(#P=0.042)and Nav1.3(#P=0.0162)and attenuated the downregulation of miR-30b(###P<0.0001).In addition,we found that mi R-30 b antagomir transfection upregulated Nav1.3 while it downregulated miR-30b(*P=0.014).The role of endogenous miR-30 b in regulating Nav1.3expression was identified in primary DRG neurons.Taken together,we demonstrated that miR-30 b suppressed the expression of Nav1.3 by binding with SCN3 A 3'UTR.(5)MiR-30 b agomir was able to attenuate neuropathic pain induced by SNL with intrathecal injection.MiR-30 b agomir delivered to SNL rats for 4 days following day 10 with intrathecal injection,and 50% PWTs and PWLs were tested.At day 10 after SNL,neuropathic pain was established(***P<0.0001).From day 2 following drug administration,the mechanical allodynia and thermal hyperalgesia caused by SNL were attenuated by intrathecal injection with miR-30 b agomir;to test whether miR-30 b agomir could repress the expression of Nav1.3,we measured the expression of miR-30 b and Nav1.3 by qRT-PCR and western-blot.MiR-30 b agomir reversed the upregulation of SCN3 A and downregulation of miR-30 b in SNL rats.In western-blot data,the upregulation of Nav1.3 protein was effectively inhibited by miR-30 b agomir in DRG neurons(*P=0.0303)and spinal cord(*P=0.0110)in SNL rats.These results confirm that miR-30 b overexpression reverses the upward tendency of Nav1.3 in SNL rats at the level of mRNA and protein,leading to a partial easement of pain.MiR-30 b was downregulated by intrathecal injection with miR-30 b antagomir in na?ve rats.We applied miR-30 b antagomir to na?ve rats for 4 days and determined their sensitivity to mechanical and thermal stimulus.We found that the threshold values for mechanical and thermal stimulus were significantly lower during miR-30 b antagomir delivery than those of na?ve rats injected with scrambled miRNAs,demonstrating that miR-30 b antagomir produced pain behaviors in na?ve rats.RT-PCR and western-blot data showed that down regulation of miR-30 b induced increases in Nav1.3 mRNA in DRG neurons(**P=0.0011)and in spinal cord(*P=0.0344),as well as protein expression(DRG,*P=0.0341;spinal cord,*P=0.0309)Research conclusion:We found that miR-30 b directly targets SCN3 A 3'UTR both in vitro and in vivo,and we demonstrated that miR-30 b alleviates neuropathic pain by suppressing the expression of Nav1.3 in DRG neurons and spinal cord following SNL.These findings indicate that miR-30 b is involved in the regulation of neuropathic pain by targeting Nav1.3,which might provide new insight into the clinical treatment of neuropathic pain. |
PDF Full Text Request