环状RNAcircRbfox1抑制miR-146a和miR-155调控MyD88/NF-κB信号转导通路参与神经病理性疼痛的机制研究

Circular RNA (circRNAs) is a kind of characterized by closed loop structure, endogenous non-coding RNA as competitive endogenous RNA targeted regulation of micrornas (ceRNA) affect the gene expression.The specific effect of most circRNAs neuropathy pain is unknown.Previous studies have found that circRbfox1 down-regulated in DRG ;RNA - FISH illustrates that circRbfox1 is located in the cytoplasm of peripheral nociceptive neurons.The result of bioinformatics analysis indicated that there are more than two binding sites between circRbfox1 and miR-146a and miR-155.In the propose project, In this study, we plan 1) To analyze whether or not in vitro silencing endogenous circRbfox1 or specific overexpression of circRbfox1 can significantly affect cell functions of nociceptive neurons;2) Via luciferase screening assay, to determine whether or not circRbfox1 can sponge to miR-146a and miR-155 with potential binding sites and directly inhibits miR-146a and miR-155 activity, and to screen and validate their potential downstream targets; 3) In vivo, to clarify the relationship between expressions of circRbfox1 and pain behavior change of neuropathic pain rats. In summary, the regulatory role of circRbfox1 in inflammatory cytokines release and peripheral sensitization involved in neuropathic pain are expectable from this project, which will definitely explode our understading of complex mechanisms of neuropathic pain and contribute to the clinical screening of targeted therapy.

神经病理性疼痛已成为目前困扰人类健康的主要疾病之一，尚无明确有效的治疗手段，阐明其发病机制寻找新的治疗策略迫在眉睫。环状RNA(circRNAs)可靶向调控miRNA的表达，参与多种疾病的发生发展过程。前期研究发现circRbfox1在DRG组织中呈低表达，且主要存在于细胞质中。生物信息学分析预测circRbfox1可能存在miR-146a和miR-155的互补结合位点，而后两者可影响MyD88/NF-κB信号通路参与神经病理性疼痛。因此，我们推测circRbfox1可能作为“海绵”靶向抑制miR-146a和miR-155，影响其下游靶基因表达，最终调控MyD88/NF-κB信号通路产生外周敏化导致神经病理性疼痛。本课题将进一步分析circRbfox1的生物学特征，从体内体外层面通过沉默或上调circRbfox1的表达进一步证明假说，为揭示神经病理性疼痛的发病机制以及靶向治疗提供新的思路。

神经病理性疼痛（NP）已成为目前困扰人类健康的主要疾病之一，其常发生于神经损伤，但神经损伤导致NP发生发展的机理目前尚不明确。有研究发现慢性疼痛可引起机体表观遗传学发生改变，而环状RNA可靶向调控miRNA的表达参与多种疾病的发生发展过程。在本研究项目中申请人发现外周神经损伤后背根神经节脱髓鞘，可引起神经元中环状RNA的差异性表达，其中细胞质中的circRbfox1显著下调。急性分离短期原代培养DRG神经元性细胞功能实验及荧光素酶报告基因检测结果证实circRbfox1与miR-146a及miR-155靶向结合并参与调控下游靶基因及相关信号的活化。申请人进一步构建腺相关病毒AVV及siRNA，并在大鼠DRG中原位注射。注射后的大鼠建立神经损伤神经病理性疼痛模型，发现过表达circRbfox1的神经损伤大鼠疼痛行为加剧，而进一步下调circRbfox1后miR-146a/miR-155调控的靶基因以及下游相关信号活化程度下调，建模大鼠疼痛行为有效缓解。这些结果提示我们，DRG痛觉神经元circRbfox1-miR-146a/miR-155构成的ceRNA网络参与神经损伤引起的神经病理性疼痛。该ceRNA网络可能成为治疗神经病理性疼痛的新靶点。