miR-330抑制脊髓后角GABAB受体功能介导胰腺癌中枢痛敏及其机制研究

The patients with pancreatic cancer suffer pain frequently and seriously, so the quality of life is decreased obviously, but there are still no effective analgesic methods because of the poor understanding of the mechanisms involved in the pain induction and maintenance. For the first time ,we discovered that miR-330, which is a kind of non-coding RNA,is up-regulated in naked mouse with pancreatic cancer pain, and there is no expression difference between pancreatic cancer model without pain hypersensitivity and na?ve naked mouse, although the role of miR-330 in pain hasn't been reported; metabotropic GABA receptor B is a kind of G-protein coupled receptor, which has played an important role in inhibition of hyperalgia and allodynia, and the gene GABBR2 for one of the GABA receptor subunits GABAB2 may be the target gene of miR-330, which indicates us that decrease of GABAB receptor expression by miR-330 may induce pain central sensitization in pancreatic cancer model. Our previous study is focused on peripheral nerves in pancreatic cancer pain, while this project will further pay attention to the spinal cord dorsal horn which regulate pain signaling from dorsal root ganglion significantly. So first, we need to prove the inhibition of GABAB function by miR-330 up-regulation is involved in pancreatic cancer related pain hypersensitiviy in spinal cord dorsal horn; and then in the cultured 293 cell line,spinal cord dorsal horn neuron or slices, combining with gene manipulation, molecular biology and electrophysiology technique, we will try to make sure that up-regulated miR-330 can decrease the expression of GABAB2 subunits, and find the regulation site on GABBR2 by miR-330, also the pre- and post- synaptic mechanism of decrease GABAB receptor function in inducing central hyperalgia. Finally, we want to test the hypothesis that decrease the function of miR-330 in spinal cord will alleviate pain sensation in naked mouse with pancreatic cancer pain. This study will improve our understanding of the pancreatic cancer pain for basic research and clinical treatment.

胰腺癌癌痛发生率高，缺乏有效治疗手段，严重影响患者生存质量。我们首次发现，在有痛觉过敏的胰腺癌裸鼠模型脊髓后角非编码RNA miR-330表达升高，它在疼痛中的作用尚未见报道。促代谢型GABAB受体有重要的抑制疼痛的功能，其GABAB2亚型的相应基因GABBR2是miR-330的可能作用位点。我们之前着重外周神经在胰腺癌痛中作用研究，本项目关注在疼痛调节中有重要作用的脊髓后角，拟先明确miR-330高表达抑制GABAB受体功能介导胰腺癌痛发生的病理过程，进一步利用培养的293细胞、原代脊神经元和急性脊髓切片，结合基因操作、分子生物学和电生理实验技术，证实miR-330抑制GABAB受体功能的作用位点，找到抑制GABAB功能诱发中枢痛敏的突触前及突触后机制，最后利用药理学结合动物行为实验，验证抑制脊髓后角miR-330功能对减轻胰腺癌痛的作用，为胰腺癌痛的机制研究和临床治疗提供新的研究思路。

胰腺癌癌痛发生率高，缺乏有效治疗手段，严重影响患者生存质量。通过基因芯片分析，我们发现在有痛觉过敏的胰腺癌裸鼠脊髓背角非编码RNA miR-330表达明显升高，它在疼痛中的作用尚未见报道。促代谢型GABAB受体有重要的抑制疼痛的功能，进一步地，我们经基因数据库筛查分析发现， GABAB受体的 B2 亚型的相应基因GABBR2可能是miR-330的调控作用位点。所以，我们在之前着重外周神经在胰腺癌痛中作用研究基础上, 本研究将利用脊髓背角神经元原代培养和急性脊髓切片，结合基因操作、分子生物学和电生理实验技术，重点关注miR-330高表达抑制脊髓背角神经元中GABAB2受体的表达及其相应功能，进而导致胰腺癌痛发生的可能机制，为胰腺癌痛的机制研究和临床治疗提供新的思路。基于以上的实验设计，我们得到了如下结果：1）miR-330 在痛觉过敏的胰腺癌裸鼠脊髓背角表达明显高于正常裸鼠；2）GABAB2受体在痛觉过敏的胰腺癌裸鼠脊髓背角表达明显低于正常裸鼠；3）GABBR2基因3'UTR区域miR-330有多个结合位点，并且，miR-330对GABAB2有直接的转录后调控；4）miR-330抑制脊髓背角GABAB2受体的表达和功能；5）过表达miR-330能够引起痛觉过敏。