MRAK009713 Regulates The P2X₃ Receptor In Rat DRG-Mediated Neuropathic Pain

Background and Objective:Neuropathic pain is notoriously difficult to treat with available analgesics.Molecular and cellular alterations in primary sensory neurons after peripheral nerve injury play important roles in the pathogenesis of neuropathic pain.We already know P2X₃ receptor is closely related to neuropathic pain.Many studies have shown that lnc RNAs has the function of regulating gene expression and aberrant lnc RNA expression is found to be associated with human nervous system diseases,but its exact mechanism of action remains unclear.This study was designed to investigate the effects and the possible mechanism of MRAK009713 on neuropathic pain mediated by P2X₃ receptor through reducing the expression of MRAK009713 in chronic constriction injury（CCI）rats by MRAK009713 siRNA and increasing the expression of MRAK009713 in normal rats by transfecting lnc RNA MRAK009713 full-length plasmid.Meanwhile,the proteins interacted with MRAK009713 were determined by RNA immunoprecipitation（RIP）.This study will provide a new method of prevention and treatment of chronic neuropathic pain.Methods:The effects of MRAK009713 siRNA on CCI rats were observed.To test the effect of MRAK009713 siRNA,rats were randomly divided into sham group（Sham）;CCI group（CCI）;CCI transfected with scramble siRNA group（CCI+NCsiRNA）and CCI transfected with MRAK009713 siRNA（CCI+MRAK009713siNA）group.On the 7th day after surgery,MRAK009713 siRNA or scramble siRNA was injected intrathecally into the rats at a dose of 0.25 OD/20μl/rat.The expression of MRAK009713 in the DRG of rats was detected by in-situ hybridization（ISH）and real-time PCR（q PCR）.The changes of the thermal withdrawal latency（TWL）and the mechanical withdrawal threshold（MWT）were observed.The effects of MRAK009713 siRNA on the expression of the P2X₃receptor were assessed by real-time PCR,western blot and immunohistochemistry.The effects of MRAK009713 siRNA on the phosphorylation of ERK1/2 were assessed by western blot.Furthermore,α,β-me ATP（10μM）-activated current in DRG neurons isolated from sham rats and CCI rats were recorded by whole-cell patch clamp.We compared the difference in the ATP（100μM）-activated currents of HEK293 cells transfected with p EGFP-C1-P2RX₃ plasmid alone with those of HEK293 cells co-transfected with the p EGFP-C1-P2RX₃plasmid and MRAK009713siRNA.The effects of MRAK009713 overexpression on control rats were detected.To test the effect of MRAK009713 overexpression,rats were randomly divided into control group（Ctrl）;control transfected with pc DNA3.1-MRAK009713 plasmid（Ctrl+MRAK009713）;control transfected with pc DNA3.1 plasmid（Ctrl+vector）.On the 7th day after observation,pc DNA3.1-MRAK009713 plasmid or pc DNA3.1plasmid was injected intrathecally into the rats at a dose of 5μg/20μl/rat.Seven days after transfection,the expression of MRAK009713 in the DRG was detected by real-time PCR.The changes of the thermal withdrawal latency（TWL）and the mechanical withdrawal threshold（MWT）were observed.The effects of MRAK009713 overexpression on the expression of P2X₃ receptor were assessed by real-time PCR,western blot and immunohistochemistry.The effect of overexpressing MRAK009713 on the phosphorylation of ERK1/2 was assessed by western blot.Furthermore,the interaction of MRAK009713 and P2X₃ receptor was investigated by RNA immunoprecipitation（RIP）and western blot.Results:ISH results indicated that MRAK009713 was localized in the cytoplasm of DRG neurons.Image analysis revealed that the IOD of MRAK009713in the CCI group was higher than that in the sham group（p<0.01）.The real-time PCR data showed that the expression of MRAK009713 in the CCI group was 3.9-fold higher than that in the sham group（p<0.01）.Seven days after transfection of MRAK009713 siRNA,compared with CCI group,the expression of MRAK009713in CCI+MRAK009713 siRNA group was downregulated（p<0.01）.On the 5^th or 7^thday after surgery,the MWT and TWL in the CCI,CCI+MRAK009713 siRNA and CCI+NC siRNA groups were lower than those in the sham group（p<0.01）.There was no significant difference among the CCI,CCI+MRAK009713 siRNA and CCI+NC siRNA groups（p>0.05）.Seven days after injection with MRAK009713 siRNA,the MWT and TWL in the CCI+MRAK009713 siRNA group were higher than those in the CCI group（p<0.01）,while the MWT and TWL in the MRAK009713 siRNA group remained lower than those in the sham group（p<0.05）.There was no significant difference between the CCI group and the CCI+NC siRNA group（p>0.05）.Real-time PCR,western blot and immunohistochemistry showed that the expressions of P2X₃ m RNA and protein in the CCI group were higher than those in the sham group（p<0.01）.Compared with the CCI group,the levels of P2X₃ m RNA and protein in CCI rats treated with MRAK009713 siRNA were notably decreased（p<0.01）.There was no significant difference between the CCI group and CCI rats treated with NC siRNA（p>0.05）.Western blot revealed that the IOD ratio of ERK1/2 toβ-actin did not differ significantly among the four groups（p>0.05）.However,the IOD ratio of p-ERK1/2 to ERK1/2 in the CCI group was 3.44-fold higher than that in the sham group（p<0.01）,and the IOD ratio of p-ERK1/2 to ERK1/2 in the CCI group treated with MRAK009713 siRNA was significantly decreased compared with that in the CCI group（p<0.01）.There was no significant difference in the ERK1/2phosphorylation level between the CCI group and the CCI rats treated with NC siRNA（p>0.05）.The results of whole-cell patch clamp showed that theα,β-me ATP-activated current in the CCI group was larger than that in the sham group（p<0.01）.The ATP-activated current in HEK293 cells transfected with p EGFP-C1-P2RX₃ plasmid alone could be blocked by 100 n M A-317491,a selective antagonist of the P2X₃receptor.The ATP-activated current in HEK293 cells transfected with the p EGFP-C1-P2RX₃ plasmid alone was larger than that in the cells co-transfected with the p EGFP-C1-P2RX₃ plasmid and MRAK009713 siRNA（p<0.01）.The effects of MRAK009713 overexpression on control rats:Seven days after MRAK009713 transfection,the expression of MRAK009713 in the DRG was detected by real-time PCR,the results indicated that the expression of MRAK009713in the control rats transfected with MRAK009713 was 2.4-fold higher than that in the control group（p<0.01）.Compared with control rats,the MWT and TWL in rats transfected with the MRAK009713 plasmid were decreased at the 2^nd day（p<0.05）and further reduced at the 4th and 7^th day（p<0.01）.There was no significant difference in the MWT and TWL between the control group and the control rats transfected with vector（p>0.05）.Real-time PCR,western blot and immunohistochemistry revealed that the expression levels of P2X₃m RNA and protein in the control rats transfected with MRAK009713 plasmid were higher than those in the control rats（p<0.01）.There was no significant difference in the expression levels of P2X₃ m RNA and protein between the control rats and the control rats transfected with vector（p>0.05）.Western blot showed that the IOD ratio of ERK1/2 toβ-actin was not significantly different among the control group,the control rats transfected with MRAK009713 plasmid and the control rats transfected with vector plasmid（p>0.05）.However,the IOD ratio of p-ERK1/2 to ERK1/2 in the control rats transfected with MRAK009713 plasmid was higher than that in the control group（p<0.01）.There was no significant difference in the phosphorylation of ERK1/2 between the control rats and rats transfected with vector plasmid（p>0.05）.To determine whether MRAK009713 interacts with the P2X₃ receptor,the immunoprecipitates were probed with an anti-P2X₃ antibody by western blot.We found that P2X₃ was significantly enriched by the co-expression of the pc DNA3.0-MRAK009713-12×MS2bs and pc DNA3.0-Flag-2×MS2 plasmids compared to the pc DNA3.0-12×MS2bs and pc DNA3.0-Flag-2×MS2 plasmids.Conclusion:Lnc RNA MRAK009713 can affect the pain transmission process of DRG P2X₃receptor-mediated chronic neuropathic pain through the regulation of P2X₃.