microRNA-29b通过调控下游靶基因HDAC4调节慢性炎性疼痛机制研究

Spinal and DRG neuronal sensitization contributed to the induction and maintenance of chronic pain. Neuronal sensitization mainly depends on the abnormal integration of membrane receptors, ion channels and intracellular signal transduction pathway mediated by modification of pain-related genes expression. However the molecular mechanisms underlying chronic pain development remain poorly understood. Serveral studies have shown that microRNAs are in chronic pain development by acting as post-transcriptional regulators of targert genes. Our studies found that microRNA-29b in Spinal and DRG was abnormally expression in a complete Freund's adjuvant-induced chronic inflammation pain model. Furthermore, intrathecal injection of microRNA-29b mimics could significantly reversed chronic pain behavior. Bioinformatic analysis and luciferase assays confirmed that histone deacetylase 4 (HDAC4) gene is one of the target mRNA of microRNA-29b.Intrathecal injection of SAHA(HDACs inhibitor) could also significantly reversed chronic pain behavior. A few studies indicate HDAC4 would regulate the acetylation level of transcription factors MEF2 and STAT. Modified MEF2 and STAT further promoted the transcription of a set of genes included pain-relate gene.In this proposal, we will utilize luciferase assays, FISH, qRT-PCR, western blot, high-throughput sequencing and lentiviral expression technology to elucidate the roles and mechanisms of miR-29b and target gene HDAC4 in chronic inflammatory pain.

脊髓背根神经节基因差异表达，引起神经元膜上受体、离子通道和胞内信号转导通路改变，导致神经元敏化是慢性疼痛发生发展的主要原因。既往研究发现miRNA调节疼痛相关的基因表达。前期研究证实CFA炎性疼痛模型大鼠中miR-29b在脊髓和背根神经节都有差异表达，信息学分析及靶标验证证实HDAC4是miR-29b的直接靶标，在体注射miR-29b模拟物和HDACs抑制济SAHA均可减轻CFA大鼠热激疼痛。推测脊髓背根神经节中的miR-29b表达发生变化，影响靶标HDAC4表达，HDAC4改变转录因子MEF2及STAT乙酰化水平，调控下游基因调节突触功能，从而影响炎性疼痛的发生发展。本研究在已有研究基础上，采用慢病毒表达、ChIP、高通量测序、RT-PCR、免疫印迹等技术，拟阐明miR-29b调控HDAC4导致的信号传递及对脊髓背根神经节突触功能的调节机制，为研发理想镇病药物提供新的思路和药物靶标。

脊髓背角基因差异表达，引起神经元膜上受体、离子通道和胞内信号转导通路改变，导致神经元敏化是慢性疼痛发生发展的主要原因。既往研究发现miRNA调节疼痛相关的基因表达。本项目通过miRNA高通量测序发现CFA炎性疼痛模型小鼠中miR-28，miR143等在脊髓背角发生了表达下调，信息学分析及靶标验证证实Nnat是miR-28的直接靶标，在体注射miR-28模拟物和干扰Nnat均可减轻CFA大鼠热激疼痛；Nnat主要有两个不同的剪接体：剪接体1和剪接体2，分别过表达这两个剪接体，发现剪接体1可直接引起小鼠的痛学过敏，而剪接体2不能起作用；miR-28是通过调控Nnat的表达，从而调节细胞内质网钙离子外流而参与疼痛。与此同时，本项目还研究另一个表达明显下调的miR-143,研究发现miR-143是通过调控Rab1基因的表达参与疼痛，与Rab1同一家族成员的Rab4是一个调控谷氨酸囊泡释放的关键因子，但是Rab1如果参与细胞生理功能仍未知，目前对Rab1的功能研究已有一定的结果，它可能是调控外泌体释放的关键因子，后继研究主要针对Rab1的功能进行重点研究。因此本项目的主要研究结果是：miR-28通过调控Nnat从而调节内质网钙离子释放参与疼痛；miR-143是通过调控Rab1基因表达参与疼痛，而对Rab1的具体功能仍在研究中。