| Trigeminal neuralgia(TN)is a common form of severe facial pain.The crude incidence rate was 52.2/100,000 in China.At present,non-steroidal anti-inflammatory analgesics used in the treatment of TN have poor therapeutic effect and great side effects.Therefore,the discovery of new therapeutic targets is of great significance to human health.The exact pathological mechanism of TN is still unknown.Gene expression and regulation is an important mechanism for neurological pain.Micro RNA(miRNA)is a class of endogenous non-coding small RNA that can play a role in posttranscriptional regulation.miRNA completely combined with the 3’-untranslated region(UTR)of mRNA,caused the target mRNA degradation and gene silence.It has been reported that miRNA can be involved in the occurrence and regulation of pain in the nervous system.Does miRNA play a role in TN? How is the mechanism? It will be the focus of this research.Objectives:(1)To clarify the expression changes and regulatory effects of miR-323-3p in TN rats.(2)To explore the epigenetic mechanism of miR-323-3p expression in TG of CCIION rats.(3)Screening and verification of the target gene of miR-323-3p.(4)To verify the involvement of miR-323-3p in TN rats by regulating Kv2.1.Methods:(1)SD rats were randomly grouped to establish a model of chronic compression injury of infraorbital nerve(CCI-ION).The mechanical pain threshold of the ipsilateral whisker pad was determined using von Frey filaments.(2)High-throughput sequencing and RT-qPCR were used to screen which miRNA might be involved in the regulation of TN rats.(3)The expression of miR-323-3p was detected by agarose gel electrophoresis,RT-qPCR,fluorescence in situ hybridization(FISH)and immunofluorescence.(4)The expression of miR-323-3p in TG of rats was up-regulated and downregulated in vivo,and the mechanical pain threshold of rats was detected by behavior.(5)The transcription factors regulating the expression of miR-323-3p were screened by the methods of truncating the promoter region of miR-323-3p,dual luciferase gene reporter assay,biological software prediction,FISH,immunofluorescence,western blot(WB),and chromatin immunoprecipitation(ChIP).(6)The histone modifications involved in the transcriptional regulation of miR-323-3p were detected by WB,ChIP,FISH,immunofluorescence,in vivo injection of siRNA and RT-qPCR.(7)Proteomic screening for prediction,immunoprecipitation(IP),doublelabelling immunofluorescence,WB,in vivo injection of siRNA,RT-qPCR and behavioral methods were used to verify the involvement of P300 in the transcriptional regulation of miR-323-3p.(8)Bioinformatics software prediction,double luciferase reporter gene assay,FISH,immunofluorescence,RT-qPCR,WB,Dil retrograde labeling,electrophysiology,up-regulation of Kv2.1 expression by lentivirus,up-regulation and down-regulation of miR-323-3p expression,and behavioral methods were used to confirm the regulation of Kv2.1 by miR-323-3p involved in TN.(1)Compared with rats in sham group,mechanical pain sensitivity in the ipsilateral whisker pad of rats in CCI-ION group was significantly relieved 14 days after surgery,and lasted until 28 days after surgery.(2)The expression of miR-323-3p in the TG of CCI-ION rats was significantly increased.(3)miR-323-3p is widely expressed in rat TG,and its expression is increased in CCI-ION rat TG,and mainly expressed in neurons.(4)The up-regulated expression of miR-323-3p in vivo resulted in the significant decrease of mechanical pain threshold in rats.The down-regulation of miR-323-3p in vivo resulted in the significant increase of mechanical pain threshold in rats.(5)The core transcription region of miR-323-3p is located in the upstream promoter region 0-438 bp.miR-323-3p colocalized with transcription factor FOXA2 in TG neurons of rats,and the expression of FOXA2 protein in TG of CCI-ION rats remained unchanged.The enrichment of FOXA2 in the promoter of miR-323-3p was increased in CCI-ION rats.(6)The protein levels of H4R3me2 a and PRMT1 increased in the TG of CCI-ION rats,and the enrichment of H4R3me2 a in the promoter region of miR-323-3p gene increased in the TG of CCI-ION rats,and there was colocalization of miR-323-3p and H4R3me2 a.After injection of PRMT1 siRNA,the expressions of PRMT1 and miR-323-3p were decreased,and the enrichment of H4R3me2 a in the promoter region of miR-323-3p gene was decreased in rat TG.(7)P300 protein expression in TG of CCI-ION rats remained unchanged.P300 colocalized with PRMT1,and P300 binding with PRMT1 increased after CCI-ION.After peripheral nerve ligation,histone H3 acetylation level increased significantly.After injection of P300 siRNA,H3 acetylation of miR-323-3p promoter region and expression of miR-323-3p were decreased,and mechanical pain threshold of rats increased significantly.Results:(8)Combined with the bioinformatics software of Target Scan and miRWalk,the target gene Kcnb1 that miR-323-3p may regulate was predicted and screened.miR-323-3p co-located with Kv2.1,and the mRNA expression of KV2.1 decreased after CCIION,and was negatively correlated with the expression of miR-323-3p.The current density of Kv2.1 channel was decreased in CCI-ION rat TG neurons.Kv2.1 protein expression,Kv2.1 current density and mechanical pain threshold of rats increased significantly after Lenti-Kv2.1-up injection.In vivo,the expression of miR-323-3p was up-regulated,the expression of Kv2.1 protein was significantly decreased,and the Kv2.1 current density of Dil-labeled small-diameter neurons was significantly decreased.In vivo,the expression of miR-323-3p was down-regulated,the expression of Kv2.1 protein was increased,and the Kv2.1 current density of Dil-labeled smalldiameter neurons was significantly increased.Conclusion:(1)The expression of miR-323-3p was increased in the TG of CCI-ION rats,the upregulation of miR-323-3p resulted in the decrease of mechanical pain threshold,and the downregulation of miR-323-3p resulted in the recovery of mechanical pain threshold.(2)PRMT1 recruited histone acetylase P300 and promoted the acetylation of histone H3 in the miR-323-3p promoter region,resulting in increased binding of FOXA2 in the miR-323-3p promoter region and increased transcription of miR-323-3p.(3)Kcnb1,as one of the target genes of miR-323-3p,is involved in the regulation of TN.(4)miR-323-3p is involved in TN by regulating Kv2.1 protein. |
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