miR-135a-5p通过调控CXCL12表达降低吗啡耐受  

miR-135a-5p alleviates morphine tolerance by targeting the regulation of CXCL12 expression

作　　者：何华美 陆巍 HE Huamei;LU Wei(College of Anesthesiology,Guizhou Medical University,Guiyang 550000;Department of Pain,Affiliated Hospital of Guizhou Medical University,Guiyang 550000,China)

机构地区：[1]贵州医科大学麻醉学院,贵州贵阳550000 [2]贵州医科大学附属医院疼痛科,贵州贵阳550000

出　　处：《基础医学与临床》2024年第5期665-672,共8页Basic and Clinical Medicine

基　　金：贵州省卫生健康委科学技术项目(gzwkj2023-395)。

摘　　要：目的研究miR-135a-5p调控CXCL12对C57BL/6J小鼠吗啡耐受的影响。方法将64只小鼠随机分为:对照(NS)组、吗啡耐受(MT)组、CXCL12沉默(CXCL12-siRNA)组、沉默阴性对照(NC-siRNA)组、miR-135a-5p激动剂(miR-agomir)组、激动剂阴性对照(miR-NC)组、miR-135a-5p激动剂+CXCL12过表达(miR-agomir+LV-CXCL12)组、miR-135a-5p激动剂+过表达阴性对照(miR-agomir+LV-control)组。通过皮下注射给药建立吗啡镇痛耐受模型,采用温水甩尾法测定各组小鼠给药前和给药后的热甩尾时间(TL);造模结束后麻醉处死小鼠并取L4~L5脊髓组织,RT-qPCR检测miR-135a-5p及CXCL12 mRNA的表达,Western blot检测CXCL12蛋白的表达,双荧光素酶报告基因实验检测miR-135a-5p、CXCL12的靶向关系。结果1)成功建立吗啡耐受小鼠模型,与NS组相比,MT组CXCL12 mRNA及蛋白表达显著升高(P<0.05),miR-135a-5p显著下调(P<0.05)。2)沉默Cxcl12及过表达miR-135a-5p均提高吗啡耐受小鼠热痛阈值,显著减缓吗啡耐受的发生发展(P<0.05)。3)与miR-NC相比,miR-agomir组CXCL12 mRNA及蛋白表达均下降(P<0.05)。4)双荧光素酶报告基因实验显示miR-135a-5p靶向作用于Cxcl12。5)过表达CXCL12可逆转miR-135a-5p对吗啡耐受小鼠的热痛保护作用。结论miR-135a-5p通过靶向抑制CXCL12表达进而缓解吗啡耐受的形成。Objective Investigating the effect of miR-135a-5p in regulating of CXCL12 on the formation of morphine tolerance in C57BL/6J mice.Methods Totally 64 mice were randomly divided into the following groups:control(NS)group,morphine tolerance(MT)group,CXCL12 inhibitor(CXCL12-siRNA)group,inhibitor negative control(NC-siRNA)group,miR-135a-5p agonist(miR-agomir)group,agonist negative control(miR-NC)group,miR-135a-5p agonist+CXCL12 overexpression(miR-agomir+LV-CXCL12)group,and miR-135a-5p agonist+CXCL12 over-expression negative control(miR-agomir+LV-control)group.Morphine tolerance models were established by subcutaneous injection.The tail-flick test was used to measure the thermal tail-flick latency(TL)before and after drug administration in each group.After modeling,the mice were euthanized under anesthesia,and L4~L5 spinal cord tissues were collected.RT-qPCR method was used to detect the expression of miR-135a-5p and CXCL12 mRNA,Western blot was used to detect the expression of CXCL12 proteins,and a dual luciferase reporter gene assay was used to detect the targeting relationship between miR-135a-5p and CXCL12.Results 1)The morphine-tolerant mouse model demonstrated a significant increase in CXCL12 mRNA and protein expression(P<0.05)and a significant downregulation of miR-135a-5p(P<0.05)compared to the NS group.2)Silencing Cxcl12 and over-expression of miR-135a-5p both increased the thermal pain threshold in morphine-tolerant mice,significantly slowed down the occurrence and progression of morphine tolerance.3)Compared to the miR-NC group,the miR-agomir group showed a decrease in both CXCL12 mRNA and protein expression(P<0.05).4)Dual-luciferase reporter gene experiments demonstrated that miR-135a-5p was targeted at Cxcl12.5)Over-expression of CXCL12 reversed the thermal pain-protective effect of miR-135a-5p in morphine-tolerant mice.Conclusions It is demonstrated by the results above that miR-135a-5p alleviates the formation of morphine tolerance by suppressing the expression of CXCL12.

关 键 词：吗啡耐受 CXC趋化因子配体12(CXCL12) 微小RNA miR-135a-5p 

分 类 号：R441.1[医药卫生—诊断学] R614[医药卫生—临床医学]