Dna Methylation Regulates Neuropathic Pain By Targeting K_2p1.1 Gene In Dorsal Root Ganglion

BackgroundsNeuropathic pain is a severe long-standing painful experience.Due to its multi-faceted etiology and incompletely-understood mechanism,there is barely successful pharmacological treatment by now and neuropathic pain has been a global medical problem.Dorsal root ganglion(DRG)contains the cell bodies of primary sensory neurons.Etiological factor can cause phenotypic changes in DRG neuron which shows hyperexcitability and increases nociceptive afferent inputs.The activated DRG neuron can lead to sensitization of central nerve system via release of glutamate and substance P.Thus,exploring the underlying changes of receptor and ion channel in DRG is essential for understanding the pathogenesis of neuropathic pain.Ion channel is the molecular basic of neuronal electric activities.Potassium ion channel(potassium channel)represents the most diverse and complex functional class of ion channel.There are four major classes of potassium channels including voltage-gated potassium channel,calcium-activated potassium channel,inwardly rectifying potassium channel and tandem pore domain potassium channel.These four major classes of potassium channels play different roles during the neuronal action potential.Tandem pore domain potassium(K2P)channel generates leak or background potassium current driving by the asymmetric K+ gradient across the membrane and is responsible for maintenance of rest membrane potential.Theoretically,the downregulation of K2 p channel can lead to a depolarization of rest membrane potential and neuronal hyperexcitability.Our previous research shows that some K2 p channels decrease in DRG after peripheral nerve injury which indicates that K2 p channels may be important in the development of neuropathic pain,although its transcriptional regulation mechanism is still unknown.Epigenetic regulation describes the phenotypic trait changes resulting from developmental or environmental factors while not due to variations in DNA sequence.Histone modification,DNA methylation and non-coding RNA are well-known epigenetic regulation.DNA methylation is the first epigenetic signal to be recognized which is involved in numerous physiological and pathophysiological process including neuronal plasticity and cancer.The research of DNA methylation in the development of neuropathic pain is still in its infancy.In this project,we focus on the role of DRG K2p1.1 in the development of neuropathic pain and whether DNA methylation participates in the transcriptional regulation of K2p1.1 and provide theoretical basis for the treatment of CINP.Methods1.To verify whether K2p1.1 is involved in the development and maintenance of neuropathic pain,we(1)establish chemotherapy and peripheral nerve injury induced neuropathic pain mice model,check the expression change of K2p1.1 at different time point with Western Blot and Real-time Quantitative PCR;(2)check the expression changes of K2p1.1 and co-localization in different type of DRG cell with immunohistochemistry methods;(3)check the behavior of mice and expression of K2p1.1 after DRG microinjection of si RNA which specifically targets and knocks down K2p1.1,and record electrophysiology changes of DRG neurons after si RNA microinjection with whole-cell patch clamp technique;(4)check the behavior changes of neuropathic pain model and expression of K2p1.1 after DRG microinjection of AAV-K2p1.1 which can overexpress K2p1.1.2.To verify whether the expression of K2p1.1 is regulated by DNA methyltransferase and contribute to the development of neuropathic pain,we(1)establish chemotherapy induced pain model and check the expression of DNMT1、DNMT3a and DNMT3 b in DRG at different time point with Western Blot and Real-time Quantitative PCR;(2)check the co-localization of DNMT3 a and K2p1.1 in DRG with double-labelling immunofluorescence;(3)check the mechanical and thermal induced pain threshold of mice after treated with chemotherapy drug and/or RG108(DNA methyltransferase inhibitor);(4)check the behavior change of DNMT3 a knock-out mice which is DRG tissue-specific KO using Advillin-Cre/Lox P system after treated with chemotherapy drug and detect the expression changes of DNMT3 a and K2p1.1;(5)construct HSV-DNMT3 a overexpression vector,culture wildtype DRG neuron and infect it with HSV-DNMT3 a,check the expression changes of DNMT3 a and K2p1.1(6)construct AAV5-Cre virus,culture DNMT3aflox/flox mice DRG neuron and infect it with AAV5-Cre,check the expression changes of DNMT3 a and K2p1.1 after acute knock-out of DNMT3 a.3.To verify the DNA methylation of KCNK1(K2p1.1 gene name)promoter carried out by DNMT3 a,we(1)design 8 pairs of PCR primers that cover the Cp G island of KCNK1,use chromatin Immunoprecipitation method to detect the binding affinity between DNMT3 a and KCNK1 promoter;(2)use methylated DNA Immunoprecipitation to check the DNA methylation level of KCNK1 promoter in DRG after treated with chemotherapy drug.Results1.After treated with chemotherapy drugs or peripheral nerve injury,DRG K2p1.1 decreases significantly in both protein and m RNA level when compared with control group.2.Using double labeling,we found that K2p1.1 co-expressed with βIII-tubulin(a specific neuronal marker),but not with GS(glutamine synthetase,a marker for satellite glial cells),indicating that K2p1.1 is expressed exclusively in the neurons of DRG.Double labeling also shows that K2p1.1 express in different type of neuron,approximately 45.8% of K2p1.1-labeled neurons are positive for NF200(a marker for myelinated medium-and large-diameter neurons),31.9% for CGRP(a marker for small unmyelinated peptidergic neurons)and 18.9 % for IB4(a marker for small unmyelinated non-peptidergic neurons).3.Three days after microinjection of si RNA to knock down DRG K2p1.1,mechanical stimulus induced withdrawal percentage of mice increased significantly and thermal stimulus induced withdrawal latency decreased significantly,which indicates that knock down DRG K2p1.1 can induced pain like behavior.Whole cell current clamp recording found that KCNK1 si RNA could significantly increase the rest membrane potential by 10.6m V,8.6m V and 7.9m V in large,medium and small DRG neurons respectively.The DRG neurons showed spontaneous activity had 24%,36% and 38% increment in large,medium and small DRG neurons respectively.4.DRG microinjection of AAV-DJ-K2p1.1 virus could block and reverse chemotherapy or peripheral nerve injury induced neuropathic pain.5.In DRG,The expression of DNMT3 a while not DNMT1 or DNMT3 b,increased significantly in chemotherapy induced neuropathic pain model.DNMT3 a was co-localized with K2p1.1 in DRG neuron.6.RG108 can reverse chemotherapy induced neuropathic pain.Similarly,chemotherapy cannot induce pain-like behavior when mice were DNMT3 a tissue-specific knocked out in DRG.It indicates that DNMT3 a is involved in the development of chemotherapy induced neuropathic pain.7.In vitro study found that overexpression of DNMT3 a can inhibit the expression of K2p1.1 in protein and m RNA level,while knock-out of DNMT3 a can increase the expression of K2p1.1 significantly.It indicates that DNMT3 a regulates the expression of K2p1.1.8.Chromatin Immunoprecipitation experiment found that DNMT3 a can bind the KCNK1 promoter and the binding affinity between DNMT3 a and KCNK1 promoter increased 3.2 folds after mice were treated with chemotherapy drug.Methylated DNA immunoprecipitation experiment found that DNA methylation level of the KCNK1 promoter region where DNMT3 a bound with increased 3.1 folds after mice was treated with chemotherapy,while this DNA methylation level decreased to 22% in DNMT3 a knock-out mice.ConclusionsThe downregulation of K2p1.1 in DRG neuron can lead to neuronal hyperexcitability and participate in the development and maintenance of neuropathic pain.In neuropathic pain model,the upregulation of DNMT3 a can increase the DNA methylation level of KCNK1 promoter and inhibit its transcriptional activity subsequently.This may play an important role in the pathogenesis of neuropathic pain.