Wnt3a信号通路通过JMJD6的表观遗传修饰参与神经病理性疼痛
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摘要
目的:探讨Wnt3a通过Jumonji C结构域6(Jumonji C domain 6,JMJD6)的表观遗传修饰在神经病理性疼痛中发挥作用的机制。方法:将SD大鼠分为4组:Sham组,慢性缩窄性损伤(chronic constriction injury,CCI)组,CCI+阴性慢病毒表达载体(LV-NC)组;CCI+慢病毒过表达载体(LV-JMJD6)组。构建SD大鼠坐骨神经CCI模型和JMJD6慢病毒表达载体。CCI术后第3天通过鞘内导管给药,按照分组分别给予生理盐水和含慢病毒的试剂(病毒滴度1×108 TU/mL)各20μL。监测大鼠的机械缩足阈值(paw withdrawal mechanical threshold,PWMT)和热缩足潜伏期(paw withdrawal thermal latency,PWTL),并运用蛋白质印迹法检测脊髓水平Wnt3a及NR2B蛋白的表达变化,免疫共沉淀检测JMJD6与Wnt3a之间是否存在直接相互作用。结果:与Sham组相比,CCI术后各组大鼠的PWMT明显降低和PWTL明显缩短(P<0.05)。与CCI组和CCI+LV-NC组相比,CCI+LV-JMJD6组的PWMT在术后第10和14天明显升高,PWTL在术后第14天明显延长(P<0.05)。CCI术后第14天,CCI组及CCI+LV-NC组Wnt3a和NR2B蛋白表达水平较Sham组明显升高,鞘内注射慢病毒载体后,CCI+LV-JMJD6组的Wnt3a和NR2B蛋白表达水平较CCI+LV-NC组降低(P<0.05)。免疫共沉淀结果显示Wnt3a与JMJD6之间无直接相互作用。结论:Wnt3a参与调节神经病理性疼痛,其作用可能与JMJD6的表观遗传修饰相关,两者可能通过间接相互作用进行调节。
Objective:To explore whether Wnt3a exerts a role in neuropathic pain through Jumonji C domain 6(JMJD6)-associated epigenetic modification.Methods:SD rats were divided into 4 groups:A sham group,a chronic constriction injury(CCI) group,a CCI+negative lentiviral expression vector(LV-NC) group and a CCI+lentiviral overexpression vector(LV-JMJD6) group.The sciatic nerve CCI model of SD rat and JMJD6 lentiviral expression vector were constructed.On the third day after CCI,the intrathecal catheter was prepared,and 20 μL of normal saline and lentivirus-containing reagent(virus titer 1×108 TU/mL) were administered.The rats' paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were monitored,and Western blotting was used to detect the expression of Wnt3a and NR2B protein in the spinal cord.Co-immunoprecipitation was applied to detect the interaction between JMJD6 and Wnt3a.Results:Compared with the sham group,the PWMT of the rats in each group after CCI was significantly decreased and the PWTL was significantly shortened(P<0.05).Compared with the CCI group and the CCI+LV-NC group,PWMT in the CCI+LV-JMJD6 group was increased significantly on the 10 th day and the 14 th day after CCI,and the PWTL was significantly prolonged on the 14 th day after CCI(P<0.05).On the 14 th day after CCI,the expression levels of Wnt3a and NR2B in the CCI group and the CCI+LV-NC group were significantly higher than those in the sham group.After intrathecal injection of lentiviral vector,Wnt3a and NR2B protein expression levels in the CCI+LV-JMJD6 group were lower compared with the CCI+LV-NC group(P<0.05).The results of co-immunoprecipitation showed no direct interaction between Wnt3a and JMJD6.Conclusion:Wnt3a is involved in mediating neuropathic pain,and its effect may be related to the epigenetic modification of JMJD6,which is likely regulated through indirect interaction.
Objective:To explore whether Wnt3a exerts a role in neuropathic pain through Jumonji C domain 6(JMJD6)-associated epigenetic modification.Methods:SD rats were divided into 4 groups:A sham group,a chronic constriction injury(CCI) group,a CCI+negative lentiviral expression vector(LV-NC) group and a CCI+lentiviral overexpression vector(LV-JMJD6) group.The sciatic nerve CCI model of SD rat and JMJD6 lentiviral expression vector were constructed.On the third day after CCI,the intrathecal catheter was prepared,and 20 μL of normal saline and lentivirus-containing reagent(virus titer 1×108 TU/mL) were administered.The rats' paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were monitored,and Western blotting was used to detect the expression of Wnt3a and NR2B protein in the spinal cord.Co-immunoprecipitation was applied to detect the interaction between JMJD6 and Wnt3a.Results:Compared with the sham group,the PWMT of the rats in each group after CCI was significantly decreased and the PWTL was significantly shortened(P<0.05).Compared with the CCI group and the CCI+LV-NC group,PWMT in the CCI+LV-JMJD6 group was increased significantly on the 10 th day and the 14 th day after CCI,and the PWTL was significantly prolonged on the 14 th day after CCI(P<0.05).On the 14 th day after CCI,the expression levels of Wnt3a and NR2B in the CCI group and the CCI+LV-NC group were significantly higher than those in the sham group.After intrathecal injection of lentiviral vector,Wnt3a and NR2B protein expression levels in the CCI+LV-JMJD6 group were lower compared with the CCI+LV-NC group(P<0.05).The results of co-immunoprecipitation showed no direct interaction between Wnt3a and JMJD6.Conclusion:Wnt3a is involved in mediating neuropathic pain,and its effect may be related to the epigenetic modification of JMJD6,which is likely regulated through indirect interaction.
引文
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[2]Kim SY,Levenson JM,Korsmeyer S,et al.Developmental regulation of Eed complex composition governs a switch in global histone modification in brain[J].J Biol Chem,2007,282(13):9962-9972.
[3]Liu W,Ma Q,Wong K,et al.Brd4 and JMJD6-associated anti-pause enhancers in regulation of transcriptional pause release[J].Cell2013,155(7):1581-1595.
[4]Poulard C,Rambaud J,Hussein N,et al.JMJD6 regulates ERalpha methylation on arginine[J].PLoS One,2014,9(2):e87982.
[5]Denk F,Mcmahon SB,Tracey I.Pain vulnerability:a neurobiological perspective[J].Nat Neurosci,2014,17(2):192-200.
[6]Peng K,Su G,Ji J,et al.Histone demethylase JMJD1A promotes colorectal cancer growth and metastasis by enhancing Wnt/betacatenin signaling[J].J Biol Chem,2018,293(27):10606-10619.
[7]Liu S,Liu YP,Huang ZJ,et al.Wnt/Ryk signaling contributes to neuropathic pain by regulating sensory neuron excitability and spinal synaptic plasticity in rats[J].Pain,2015,156(12):2572-2584.
[8]Bennett GJ,Xie YK.A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[J].Pain1988,33(1):87-107.
[9]Milligan ED,Hinde JL,Mehmert KK,et al.A method for increasing the viability of the external portion of lumbar catheters placed in the spinal subarachnoid space of rats[J].J Neurosci Methods,199990(1):81-86.
[10]Liang L,Lutz BM,Bekker A,et al.Epigenetic regulation of chronic pain[J].Epigenomics,2015,7(2):235-245.
[11]Zhou CH,Zhang MX,Zhou SS,et al.SIRT1 attenuates neuropathic pain by epigenetic regulation of mGluR1/5 expressions in type 2diabetic rats[J].Pain,2017,158(1):130-139.
[12]Ciampi De Andrade D,Maschietto M,Galhardoni R,et al Epigenetics insights into chronic pain:DNA hypomethylation in fibromyalgia-a controlled pilot-study[J].Pain,2017,158(8):1473-1480.
[13]Mo C,Xu M,Wen C,et al.Normalizing JMJD6 expression in rat spinal dorsal horn alleviates hyperalgesia following chronic constriction injury[J].Front Neurosci,2018,12:542.
[14]Nanki K,Toshimitsu K,Takano A,et al.Divergent routes toward Wnt and R-spondin niche independency during human gastric carcinogenesis[J].Cell,2018,174(4):856-869.
[15]Tammela T,Sanchez-Rivera FJ,Cetinbas NM,et al.A Wntproducing niche drives proliferative potential and progression in lung adenocarcinoma[J].Nature,2017,545(7654):355-359.
[16]Zhang YK,Huang ZJ,Liu S,et al.WNT signaling underlies the pathogenesis of neuropathic pain in rodents[J].J Clin Invest,2013123(5):2268-2286.
[17]Yakulov T,Raggioli A,Franz H,et al.Wnt3a-dependent and-independent protein interaction networks of chromatin-bound betacatenin in mouse embryonic stem cells[J].Mol Cell Proteomics2013,12(7):1980-1994.
[18]Choi SH,Estaras C,Moresco JJ,et al.alpha-Catenin interacts with APC to regulate beta-catenin proteolysis and transcriptional repression of Wnt target genes[J].Genes Dev,2013,27(22):2473-2488.