Effects Of LncRNA Uc.48+ On Trigeminal Neuralgia

Background and Objective:Trigeminal neuralgia（TN）is the most common facial nerve pain,and there is no ideal cure.P2X₇ receptor is a unique subtype of ATP-gated ion channel family,and may be involved in the modulation and conduction of pain information.CGRP is a neuropeptide that transmits pain information and is related to the pathogenesis of trigeminal neuralgia.Long non-coding RNA（lnc RNA）involved in a variety of human disease gene regulation,and its expression changes may be associated with neurological disease.This study was designed to use lnc RNA uc.48+si RNA to interfere with the expression of uc.48+in rats with trigeminal neuralgia induced by the chronic constriction injury of the infarorbital nerve（ION-CCI）.Moreover,by transfection of uc.48+plasmid into normal rats to overexpression uc.48+,the effects of uc.48+on trigeminal neuralgia and its possible mechanism were observed.At the same time through RIP（RNA Binding Protein Immunoprecipitation,RNA-binding protein immunoprecipitation）technology,the uc.48+interaction of protein was studied,in order to provide a new method for the prevention and treatment of trigeminal neuralgiaMethods:1.Lnc RNA uc.48+si RNA experiments:The rats were randomly divided into sham operation group（Sham）,trigeminal neuralgia model group（TN）,trigeminal neuralgia+uc.48+small interfering treatment group（TN+uc.48+si RNA）,trigeminal neuralgia+uc.48+small interfering negative treatment group（TN+scramble si RNA）.On day 14 after ION-CCI operation,uc.48+si RNA or scramble si RNA was injected via infraorbitalfossa to trigeminal ganglia（TG）.The change of each rat facial mechanical withdrawal threshold（MWT）was measured by electronic tachymeter at day1,day3 and day5 after injection.The expressions of uc.48+,P2X₇and CGRP in TG were detected by q PCR and Western blotting.The expression of P2X₇ and CGRP in TG were detected by immunohistochemistry,and the co-expression of P2X₇ and GFAP（a marker of glial cell）in TG was detected by immunofluorescence double labeling.The level of serum inflammatory cytokine interleukin 1β（1L-1β）was measured by ELISA.The effect of uc.48+si RNA on the phosphorylation of ERK1/2 of TG was detected by Western blotting.2.Transfection of lnc RNA uc.48+plasmid to overexpression uc.48+experiments:the rats were randomly divided into control group（control）,transfected pc DNA3.1-uc.48+plasmid experimental group（Control+uc.48+）,and transfected pc DNA3.1 negative control group（Control+vector）.Pc DNA3.1-uc.48+or pc DNA3.1 no-load plasmid was injected via infraorbitalfossa.The change of each rat facial mechanical withdrawal threshold（MWT）was measured by electronic tachymeter at day1,day3 and day5 after injection.The expressions of uc.48+,P2X₇and CGRP in TG were detected by q PCR and Western blotting.The expression of P2X₇ and CGRP in TG were detected by immunohistochemistry,and the co-expression of P2X₇ and GFAP in TG was detected by immunofluorescence double labeling.The level of serum inflammatory cytokine 1L-1βwas measured by ELISA.The effect of lnc RNA uc.48+overexpression on the phosphorylation of ERK1/2 of TG was detected by Western blotting.3.In addition,RNA-binding protein immunoprecipitation（RIP）was used to detect the interaction between uc.48+and P2X₇ receptors.Results:1.Results of lnc RNA uc.48+si RNA experiments:Before injection of small interferon,the MWTs in TN group,TN+uc.48+si RNA group and TN+scramble si RNA group were significantly lower than in Sham group（p<0.01）,which indicating that the TN model was prepared successfully.After injection of uc.48+si RNA,from day 3 to day 5 the MWT in TN+uc.48+si RNA group was significantly increased,compared with TN group（p<0.01）,while there was no significant difference between TN group and TN+scramble si RNA group（p>0.05）.The m RNA expression of uc.48+in TN+uc.48+si RNA group was significantly lower than in TN group after transfection with uc.48+si RNA for five days（p<0.05）,indicating that uc.48+si RNA interference was performed successfully.The results of q PCR and Western blotting showed that the expression levels of P2X₇,CGRP m RNA and protein were significantly increased in TN group and TN+scramble si RNA group compared with Sham group（P<0.01）,but in TN+uc.48+si RNA group the expression was significantly lower than in TN group（p<0.05）,while between TN group and TN+scramble si RNA Group there was no difference（p>0.05）.The results of immunohistochemistry showed that the expression of P2X₇ and CGRP in TN+uc.48+si RNA group was significantly lower than in TN group（P<0.01）,but there was no significant difference between TN group and TN+scramble si RNA group（p>0.05）.Immunofluorescence double labeling method showed that there were co-expression of P2X₇ receptor and GFAP in trigeminal ganglion glial cells,the expression of P2X₇ in TN group and TN+scramble si RNA group was significantly increased compared with Sham group.In TN+uc.48+si RNA group,the co-expression was significantly lower than in TN group after treatment with uc.48+small interference.The results of ELISA showed that the level of IL-1βin TN group and TN+scramble si RNA group was significantly higher than in Sham group（P<0.01）,but in TN+uc.48+si RNA group,the level was significantly lower than in TN group（P<0.01）.The level of ERK1/2 phosphorylation in TG was measured by Western blotting,and the results showed that the integral optical density ratio of ERK1/2 toβ-actin in each group showed no difference（p>0.05）.The phosphorylation of ERK1/2 in TN and TN+scramble si RNA group was significantly enhanced compared to Sham group（P<0.05）.While in TN+uc.48+si RNA group the phosphorylation of ERK1/2 was significantly decreased compared to TN group after treatment with uc.48+small interference（P<0.05）.2.Results of transfection of lnc RNA uc.48+plasmid to overexpression uc.48+experiments:Before injection of uc.48+plasmid,the MWTs of control,control+vector and control+uc.48+group showed no significant difference.After injection of uc.48+plasmid,the MWT of control+uc.48+group was significantly lower than the control group（p<0.01）,and there was no difference between the control group and control+vector group（p>0.05）.The m RNA expression of uc.48+in the control+uc.48+group was significantly higher than in control group after transfection with uc.48+plasmid for five days（p<0.01）.,While there was no difference between the control group and control+vector group（p>0.05）,indicating uc.48+plasmid transfection was performed successfully.The results of q PCR and Western blotting showed that the expression of P2X₇,CGRP m RNA and protein in the control+uc.48+group was significantly higher than in control group（p<0.05）,and there was no difference between the control group and control+vector group（p>0.05）.The results of immunohistochemistry showed that the expression of P2X₇ and CGRP in the control+uc.48+group was significantly higher than in the control group（p<0.01）,and there was no difference between the control group and control+vector group（p>0.05）.The results of immunofluorescence double labeling method showed that there were co-expression of P2X₇ receptor and GFAP in TG glial cells,and the co-expression of P2X₇ and GFAP in the control+uc.48+group was significantly higher than in the control group.The results of ELISA showed that the level of IL-1βin the control+uc.48+group was significantly higher than in the control group（p<0.01）,and there was no difference between the control group and control+vector group（p>0.05）.The level of ERK1/2 phosphorylation in TG was measured by Western blotting,and the results showed that the integral optical density ratio of ERK1/2 toβ-actin in each group showed no statistically significant difference（p>0.05）,but the phosphorylation of ERK1/2 of TG in the control+uc.48+group was significantly higher than in the control group（P<0.01）,and there was no difference between the control group and control+vector group（p>0.05）.3.The results of RIP showed:P2X₇ was significantly enriched in the experimental groups of pc DNA3.1-uc.48+-12×MS2bs and pc DNA3.0-Flag-2×MS2 co-transfection of plasmids compared with the control group of pc DNA3.1-12×MS2bs and pc DNA3.0-Flag-2×MS2 co-transfection of plasmids,which indicating that the P2X₇receptor could specifically bind to uc.48+.Conclusion:Lnc RNA uc.48+si RNA could inhibit the pain transmission of TN,while over expression of uc.48+could facilitate the pain formation.Its mechanism might be as follows:uc.48+could promote the expression of P2X₇ and CGRP in TG,and increase the level of IL-1β,furthermore enhance the phosphorylation of ERK1/2of TG,thus affect the pain transmission of TN.And the use of uc.48+small interference can inhibit the corresponding expression,play a role in relieving pain.