Regulation Of ?7nAchR By MiR-98-5p In Rat Hypothalamus Arcuate Nucleus Of In Trigeminal Neuralgia

Objective:In the present study,we investigated the expression of miR-98-5p and?7nAchR in ARC of CCI-ION rats and analyzed the correlation between of them.Meanwhile,we explored how miR-98-5p is regulated by histone modification and how it is involved in trigeminal neuralgia by targeting ?7nAchR,which may offer new therapy target for trigeminal neuralgia.Methods:(1)The pain model of trigeminal neuralgia of rat was established by the chronic constrictive injury to the infraorbital nerve(CCI-ION).The mechanical pain threshold was measured by von Frey test in the whisker pad at 7 d,14 d,21 d,28 d.(2)Western blot analysis was applied to detect the expression of ?7nAchR in the ARC of CCI-ION rats.Immunofluorescence was used to determine the cellular distribution of ?7nAchR in ARC.(3)The ?7nAchR agonist PNU282987 was injected into ARC to detect behavioral changes of CCI-ION rats.(4)Targetscan software was used to determine the binding of miRNA with?7nAchR mRNA.PCR was applied to detect whether miR-98-5p was expressed in ARC of rats.Fluorescence in situ hybridization(FISH)was used to detect the colocalization of miR-98-5p with ?7nAchR.(5)Quantitative PCR(qPCR)was used to determine the expression level of miR-98-5p and ?7nAchR.Dual-luciferase reporter assays were applied to estimate whether miR-98-5p actually targeted ?7nAchR mRNA.(6)The miR-98-5p antagomir or miR-98-5p agomir was injected into ARC to change the content of miR-98-5p,and then corresponding behavioral changes and protein expression of ?7nAchR were detected.(7)Western blot analysis was applied to detect the acetylation of histone H3K18 in ARC of CCI-ION rats.Chromatin immunoprecipitation(ChIP)was adopted to measure the binding of histone H3K18 with the miR-98-5p gene promoter region.Result:(1)The mechanical pain of threshold of CCI-ION rats significantly reduced(p<0.001)compared with the sham groups at 14 d,21 d,and 28 d respectively.(2)The expression of ?7nAchR in ARC of CCI-ION rats was significantly decreased(p<0.05)compared with the sham groups.Injection of the ?7nAchR agonist PNU282987(1 ?L,0.1 mM)into ARC alleviated the mechanical hyperalgesia of CCI-ION rats(p<0.05).(3)The ?7nAchR was predominantly expressed in ARC neurons,but rarely with microglia as well as astrocytes.(4)Targetscan bioinformatics data showed that miR-98-5p targeted ?7nAchR mRNA.The miR-98-5p was co-localized with ?7nAchR in rat ARC.Expression of miR-98-5p was robustly increased in ARC of CCI-ION rats,while the expression level of ?7nAchR mRNA was decreased.The linear correlation analysis revealed that the downregulation of ?7nAchR was inversely related with miR-98-5p.Furthermore,dual-luciferase reporter assay showed that the miR-98-5p bound with ?7nAchR mRNA.(5)Injection of miR-98-5p antagomir into ARC alleviated the mechanical hyperalgesia of CCI-ION rats(p<0.01)and increased the expression of ?7nAchR(p<0.05).Injection of miR-98-5p agomir into ARC of naive rats induced the mechanical hyperalgesia(p<0.05)and decreased the expression of ?7nAchR(p<0.01).(6)The expression of histone H3K18ac in ARC of CCI-ION rats was significantly increased(p<0.05)compared with the sham groups.The binding of histone H3K18 with the miR-98-5p gene promoter region was enhanced in ARC of CCI-ION rats(p<0.05),suggesting that histone acetylation contributed to miR-98-5p expression.Conclusions:Histone acetylation regulated-miR-98-5p is involved in trigeminal neuralgia by negatively targeting ?7nAchR.The results suggested that miR-98-5p can be used as a new target for trigeminal neuralgia.