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1d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 C57BU6J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.

abstractsObjective 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±l.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 posttranscriptional 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.