国际麻醉学与复苏杂志   2017, Issue (8): 4-4
    
microRNA-93靶向调控Rho相关激酶2/LIMK/Cofilin 通路影响骨癌痛小鼠痛行为
张旭东, 任鹏程, 何印斌, 孙丽, 刘九虎1()
1.第四军医大学唐都医院
Study on promotion of bone cancer pain via rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling by negative regulation of microRNA in mice
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摘要：

目的　观察microRNA-93（miR-93）及Rho相关激酶（rho associated coiled-coil forming protein kinase, ROCK）通路对小鼠骨癌痛（bone cancer pain， BCP）行为的影响并探讨其潜在机制。　方法　将C57BL/6J小鼠采用随机数字表法分为对照组（Control组）、假手术组（Sham组）及BCP组、BCP+PBS组、BCP+Y-27632（ROCK抑制剂）组，每组13只。建模前1 d及建模后3、5、7、10、14 d进行动物痛行为学评估，观察小鼠对机械和热刺激的反应。建模后第14 天处死小鼠，取术侧L4~L6 背根神经节（dorsal root ganglion， DRG），SYBR Green实时荧光定量PCR法观察miR-93表达变化，双荧光素酶实验检测miR-93对ROCK2 基因的靶向调控。Western blot法观察ROCK2、LIMK（LIM domain kinase, LIMK）和Cofilin蛋白在DRG神经元中表达。　结果　C57BL/6J小鼠股骨接种NCTC2472 细胞后，患侧后肢分别在第5、7天开始出现明显的触诱发痛［Sham组（6.81±1.04） g，BCP组（5.12±0.47） g］和热痛过敏［Sham组（11.75±1.46） s，BCP组（7.61±1.07） s］，并呈进行性加重（P<0.05）；BCP组小鼠miR-93表达随疼痛加重呈时间依赖性降低，而ROCK2表达呈时间依赖性增加（P<0.05），进而激活LIMK/Cofilin通路。双荧光素酶实验证实miR-93可与ROCK2 mRNA 3'-UTR直接结合，从而发挥对ROCK2 转录后翻译的抑制作用（P<0.05）。鞘内注射ROCK抑制剂能有效缓解小鼠触诱发痛［BCP+PBS组（3.43±0.89） g，BCP+Y-27632组（5.74±1.02） g］和热痛过敏［BCP+PBS组（5.48±1.03） s，BCP+Y-27632组（9.82±1.24） s］，同时抑制通路主要因子ROCK2、磷酸化-LIMK（phospho-LIMK， p-LIMK）、磷酸化-Cofilin（phospho-Cofilin， p-Cofilin）的表达（P<0.05）。　结论　小鼠BCP疼痛过程中miR-93表达降低，进而激活ROCK2/LIMK/Cofilin 通路，鞘内注射ROCK抑制剂可抑制该通路的活化，缓解小鼠BCP症状。
关键词： 骨癌痛；　镇痛；　microRNA-93；　Rho相关激酶2/LIMK/Cof
Abstract:

Ｏｂｊｅｃｔｉｖｅ　To observe microRNA (miR-93) regulation in bone cancer pain(BCP) and explore the underlying mechanisms of miR-93 and Rho associated coiled-coil forming protein kinase (ROCK) signaling affecting the biological behaviors of BCP in mice.　Methods　Male mice were randomly divided into Control group, Sham group, bone cancer pain (BCP group), BCP+PBS group and BCP+Y-27632(ROCK inhibitor) group(n=13). The paw mechanical threshold and paw thermal latency were used to evaluate the behavioral changes on 1 day before surgery and 1, 3, 5, 7, 10, 14 after surgery. L4-L6 dorsal root ganglions were removed on postoperative day 14, followed by Real-time PCR to measure the expression of miR-93. The ROCK2 promoter activity regulated by miR-93 was evaluated by a dual luciferase reporter assay. Western blotting was used to evaluate the protein expression level of ROCK2, phospho-LIM domain kinase (LIMK) and phospho-Cofilin.　Results　Following intrafemoral carcinoma inoculation, robust mechanical allodynia ［Sham group(6.81±1.04) g, BCP group(5.12±0.47) g］and thermal hyperalgesia ［Sham group(11.75±1.46) s,BCP group (7.61±1.07) s］ in C57BL/6J mice were respectively developed on day 5 and 7, and the symptoms grow progressively with time (P<0.05). The expression level miR-93 is decreased in a time dependent way in BCP group with progressively increased pain. However, the ROCK2 expression level was increased (P<0.05), and subsequently activated the LIMK/Cofilin pathways. Dual luciferase reporter assay demonstrated that miR-93 directly targeted the 3'-UTR of the ROCK2 gene, resulting in inhibition of the post-transcriptional translation of ROCK2． ROCK inhibitor significantly up-regulated paw withdrawal threshold ［BCP+PBS group(3.43±0.89) g, BCP+Y-27632 group (5.74±1.02) g］, paw withdrawal latency ［BCP+PBS group (5.48±1.03) s, BCP+Y-27632 group (9.82±1.24) s］ and reduced the protein expression level of ROCK2, phospho-LIMK and phospho-Cofilin (P<0.05).　Conclusions　Reduced expression of miR-93 can promote BCP through activating ROCK2/LIMK/cofilin pathway. Therefore, miR-93 or ROCK inhibitor may be novel targets for BCP.
Key words: Bone cancer pain;　Analgesia;　MicroRNA-93;　Rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling