Functional Regulation And Mechanisms Of T-type Calcium Channel Cav3.2 In Trigeminal Neuralgia

Trigeminal neuralgia(TN)is a common cranial nerve disease,characterized by severe unilateral paroxysmal and refractory pain in the region of the trigeminal nerve.At present,its etiology and pathogenesis are still largely not clear and the treatments are not satisfactory using drugs in clinical.The characteristic biophysical features of T-type calcium channels,including activation with small membrane depolarization,relatively fast and voltage-dependent inactivation kinetics and slow deactivation kinetics upon closing the channel,make them very suitable for controlling cellular excitability of sensory neurons in peripheral and central nervous system.Recently,studies have shown that the expression and functional changes of t-type calcium channels are involved in the development and maintenace of neuropathic pain and can serve as a target for pain therapy.Three a1-subunit genes,CACNA1 G,CACNA1H,and CACNA1 I are known to encode a1G(Cav3.1),a1H(Cav3.2)and a1I(Cav3.3)isoforms of the T-type calcium channel family,respectively.Among them,Cav3.2 plays a pivotal role in nociceptive processing in neuropathic pain.However,the alteration of Cav3.2 expression and function in trigeminal neuralgia and the underlying mechanisms has not been fully elucidated.The mature mi RNA binds to the 3’-untranslated region(3’-UTR)of target m RNA and represses translation,which play an important role in regulation of protein expression at post-transcription level.There are increasing evidences that mi RNAs are involved in pain regulation.In the TG of trigeminal neuralgia,it remains unknown whether Cav3.2 expression is regulated by mi RNAs and which mi RNA is involved.Here,we aim to investigate the modulation of Cav3.2 expression by mi RNA and the role of histone modification in mi RNA transcription,which will provide certain theoretical basis for clinical treatment of trigeminal neuralgia.Objectives:(1)To detect the expression and involvement of Cav3.2 in trigeminal neuralgia.(2)To screen and verify of the regulation of mi R-32 on Cav3.2 expression.(3)To clarify mi R-32 regulating the expression of Cav3.2 and participating in regulating trigeminal neuralgia.(4)To investigate the modification of histone methylation in mi R-32 expression in TG of trigeminal neuralgia rats.Methods:(1)Adult male SD rats were used to establish chronic constrictive injury of the inferior orbital nerve(CCI-ION)models,and the mechanical pain threshold of the whiskers pad was stimulated by von Frey filaments.(2)The changes of Cav3.2 expression in the trigeminal ganglion(TG)of rats were detected using the western blot(WB)method.The distribution of Cav3.2 in NF-200,CGRP and IB4 positive neurons was observed by immunofluorescence staining.(3)The cell bodies of inferior orbital nerve were retrograde labeled by Di I,a kind of cell membrane fluorescent dye.Whole-cell patches clamp technique was applied to detect the current density of T-type calcium channel in small diameter neurons labeled by Di I,in the TG of the CCI-ION or sham rats.(4)The inhibitor of T-type calcium channel,Z941 was applied,combining with behavioral and electrophysiological methods to verify the involvement of T-type calcium channel in hyperalgesia.(5)The bioinformatics softwares of micro RNA.org and Targetscan.org were used to screen the mi RNA that may regulate Cav3.2 expression,namely mi R-32.(6)The fluorescence in situ hybridization(FISH),real-time fluorescence quantitative PCR(RT-q PCR)and dural luciferase report gene assay were used to validate the role of mi R-32 in regulating Cav3.2 expression.(7)The regulations of mi R-32 in Cav3.2 in vivo were detected by overexpression or inhibition of mi R-32.(8)WB,FISH and chromatin immuneprecipitation(Ch IP)methods were adopted to detect the enrichment of histone H3 ninth lysine dimethylation(H3K9me2)and histone H3 27 th lysine trimethylation(H3K27me3)in mi R-32 promoter region.(9)The inhibitors of methylation of H3K9me2 and H3K27me3 were used to evaluation the role of histone methylation in mi R-32 expression and animal pain behavior.Results:(1)The mechanical pain threshold of rats was significantly decreased in the 14 d after CCI-ION,and the rats in the 21 d and 28 d continued to suffer from hyperalgesia.(2)CCI-ION induced an upregulation of Cav3.2 expression in TG.However,there was no significant alteration in the expression levels of Cav3.1 and Cav3.3 at different points after CCI-ION.(3)Cav3.2 was mainly distributed in the medium-small diameter neurons of CGRP and IB4 markers,which were associated with pain.(4)CCI-ION increased the current density of T-type calcium channel significantly in Di I labeled small diameter neurons.Z941,an inhibitor of T-type calcium channels,antagonized this increase of current density,as well as alleviating the mechanical hyperalgesia.(5)Biological information software analysis suggested that Cav3.2 m RNA might be a target of mi R-32,which expressed extensively in TG and co-localized with Cav3.2.(6)The expression of mi R-32 was down-regulated in the TG of CCI-ION rat.(7)Dural luciferase report assay confirmed that mi R-32 could regulate Cav3.2 expression.(8)Over-expression of mi R-32 down-regulated the Cav3.2 expression as well as the current density of t-type calcium channel,and alleviate the mechanical hyperalgesia.(9)When the role of mi R-32 was inhibited,Cav3.2 expression and the current density were up-regulated,causing a significant reduction in mechanical pain threshold.(10)CCI-ION induced a significant upregulation of H3K9me2 and H3K27me3 expression in TG.The cells expressed high density of H3K9me2 and H3K27me3,accompanied with low density of mi R-32,high density of Cav3.2 in the TG.(11)The results of Ch IP-q PCR indicated CCI-ION increased the enrichment of H3K27me3 and H3K9me2 in the promoters of mi R-32.(12)GSK503,an inhibitor of H3K27 methylation,decreased the level of H3K27me3,increased mi R-32 expression,and alleviated the mechanical hyperalgesia of rats.(13)UNC0638,which could inhibit H3K9me2 level,played a role in the increasing of mi R-32 level,as well as,relieved the hyperalgesia of rats.(14)The analgesic effect was more significant and the mi R-32 level was further elevated by combination of GSK503 and UNC0638.Conclusions:(1)CCI-ION induced a sustainable increasing in the Cav3.2 expression,which was involved in trigeminal neuralgia.(2)The expression of Cav3.2 was regulated by mi R-32,which was decreased in the TG of the CCI-ION rat.(3)H3K27me3 and H3K9me2 in mi R-32 promoter region regulated mi R-32 gene expression.(4)The results for the first time revealed the modification of mi RNA by histone methylation regulated the expression of Cav3.2 in neuropathic pain,which would shed new light on Cav3.2 and mi RNA as pain therapeutic targets.