类似mRNA的长非编码RNA2Y对初级感觉神经节P2X3受体介导神经病理痛的作用及机制研究

Non-coding RNAs (ncRNAs) like coding RNA play crucial roles in the control of gene expression. Long noncoding RNAs (lncRNAs) are the RNA in length of more than 200 nt, which exist stably in cells. Messenger-like long noncoding RNAs (mlncRNAs) and microRNA (in length of less than 50 nt) all are ncRNAs. Differentiated from microRNA, mlncRNAs are spliced and contain poly A tail of 3' ends and no typical open reading frame. Changes of expression and regulating factor in mlncRNAs are related with diseases, but its mechanism is unknown. Chronic pain results in agony to human. Because the mechanism of chronic pain is complex, the treatments of chronic pain are tough problem in the clinical. Our previous studies showed that long mlncRNAs (>200 nt) in rat primary sensory ganglia have been identified by transcriptome database verification and sequence predication screening of SOLiD highthroughput. On the groundwork of previous studies, we select a mlncRNA (preliminary name as mlncRNA2Y) of upregulated expression in the dorsal root ganglia of the neuropatic pain rats. In this work, the effect of mlncRNA2Y on the neuropathic pain mediated by P3X3 receptor of the dorsal root ganglia and its mechanism will be observed by the experiments of systemic and cellular levels to transfect into ganglion neurons in vitro and in vivo. The study will be beneficial to the utilization of mlncRNAs in the prevention and cure of neuropatic pain. The findings from this study will provide the knowledge for developing novel strategy to prevent and treat neuropathic pain.

非编码RNA以RNA形式在调节基因表达中起着极为重要的作用。lncRNAs一般是指大于200 nt的RNA，它们稳定存在于细胞。类似mRNA的长非编码RNA（mlncRNAs）与microRNA均是非编码RNA，但有明显区别。研究提示mlncRNAs表达的变化和影响mlncRNAs的因素改变与疾病有关，但作用机制尚不清楚。慢性疼痛给人们造成极大痛苦，由于慢性痛机理复杂，其治疗成为临床上的难题。本课题在前期SOLiD高通量转录组数据库验证和序列预测基础上选择了一个在神经病理痛大鼠初级感觉神经节（背根神经节，DRG）表达明显增加并大于200 nt的mlncRNA2Y，通过整体、细胞水平将其siRNA转染细胞后进行实验研究，观察mlncRNA2Y在DRG P2X3受体介导神经病理痛的作用及可能机制，为mlncRNAs用于神经病理痛的预防和治疗提供新的实验依据和理论基础，为慢性痛的防治探索新方法。

神经病理痛常给患者带来极大的痛苦，由于其发病机理复杂，其治疗成为临床上的难题。本项目观察长非编码RNA MRAK009713对初级感觉神经节P2X3受体介导神经病理痛的作用及机制研究。1.实验结果显示神经病理痛大鼠背根神经节MRAK009713、P2X3受体mRNA和蛋白及ERK1/2磷酸化水平明显增加，大鼠机械缩足反应阈值和热缩足反应潜伏期明显降低，DRG神经元ATP激活电流增加。2. 神经病理痛大鼠鞘内注射MRAK009713小干扰RNA后MRAK009713、P2X3受体mRNA和蛋白及ERK1/2磷酸化水平显著降低，大鼠机械缩足反应阈值和热缩足反应潜伏期明显升高，单独转染P2RX3质粒的HEK293细胞可记录到较大的ATP激活电流，而共转染MRAK009713 siRNA后ATP激活电流显著降低，其效果与P2X3受体选择性拮抗剂A-317491相似。3. 正常大鼠过表达MRAK009713后可出现类似于神经病理痛的病理变化，MRAK009713、P2X3受体mRNA和蛋白及ERK1/2磷酸化水平明显增加，大鼠机械缩足反应阈值和热缩足反应潜伏期明显降低。4. 缓激肽炎性细胞模型结果显示缓激肽处理后的DRG神经元MRAK009713及P2X3受体表达水平显著升高，细胞培养上清液中TNF-α和IL-6及细胞内[Ca2+]明显增加，MRAK009713 siRNA可明显降低MRAK009713及P2X3受体表达水平、TNF-α和IL-6及细胞内[Ca2+]的浓度，而正常DRG过表达MRAK00971后MRAK009713及P2X3受体表达水平、TNF-α和IL-6及细胞内[Ca2+]的浓度均明显升高。5. RNA-蛋白质免疫共沉淀结果显示MRAK009713与P2X3受体蛋白有相互作用。. 结果表明MRAK009713小干扰RNA可缓解神经病理痛的病理变化，其可能机制是通过与其发生相互作用的P2X3受体而减少炎性因子的释放和细胞内游离钙离子浓度，并降低ERK1/2磷酸化水平发挥作用，从而为防治神经病理痛探索新方法，并为嘌呤受体研究发现新的特异拮抗方式。