c-Jun靶向调控miR-182-5p转录在偏瘫型脑瘫上肢功能康复中的机制研究  

Mechanism of c-Jun-targeted regulation of miR-182-5p transcription in upper limb functional rehabilitation of hemiplegic cerebral palsy

作　　者：王优 徐韵贤 伍启泓 蔡凯茵 吴霖林 刘力茹 徐开寿 唐红梅 WANG You;XU Yun-xian;WU Qi-hong;CAI Kai-yin;WU Lin-lin;LIU Li-ru;XU Kai-shou;TANG Hong-mei(Department of Rehabilitation,Guangzhou Women and Children's Medical Center,Guangzhou Medical University,Guangzhou 512120,Guangdong,China;不详)

机构地区：[1]广州医科大学附属妇女儿童医疗中心康复科,广东广州512120 [2]佛山市第一人民医院康复科,广东佛山528000 [3]广州医科大学儿科学院,广东广州511436

出　　处：《广东医学》2025年第3期399-406,共8页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(82472598,81902309);广东省自然科学基金资助项目(2024A1515012920,2021A1515012543,2021A1515011303);广州市科技计划项目(2024A03J1103,2023A03J0919);广州市基础研究计划项目(市校[院]企联合资助专题)(2060206-328);广州地区临床特色技术项目(2023C-TS59)。

摘　　要：目的探讨转录因子c-Jun能否靶向结合miR-182-5p,解析其靶向结合的具体位点,并探讨强制性运动疗法(CIMT)能否通过促进c-Jun/miR-182-5p转录以提高偏瘫型脑瘫(HCP)小鼠上肢运动功能。方法慢病毒转染技术构建c-Jun敲低及过表达的Neuro-2a细胞并建立稳转株。蛋白免疫印记(Western blot)验证各组细胞c-Jun蛋白的表达情况,实时荧光定量聚合酶链式反应(RT-qPCR)验证miR-182-5p的表达情况;染色质免疫共沉淀(ChIP)验证c-Jun与miR-182-5p的结合位点。随后,构建HCP小鼠模型,予以持续2周的CIMT干预。行为学试验评估CIMT的干预效果,RT-qPCR实验验证各组小鼠左侧大脑运动皮层M1区c-Jun、miR-182-5p的表达情况。结果携带小干扰RNA(siRNA)的c-Jun敲低慢病毒si3(病毒滴度:5E+8 TU/mL)转染Neuro-2a细胞后,c-Jun蛋白表达明显降低(P<0.01);而c-Jun过表达(OE)病毒OE(病毒滴度:5E+8 TU/mL)转染Neuro-2a细胞后,c-Jun蛋白的表达显著升高(P<0.001)。miR-182-5p的转录随着c-Jun表达的减少而显著下降(P<0.01);反之,miR-182-5p转录则随之显著增加(P≤0.001),两者呈现协同变化趋势。进一步研究发现c-Jun可与miR-182-5p启动子上游-1000~-1 bp片段中多个位点结合,其中以Site 2(-71~-65 bp)结合最为明显(P<0.001)。动物行为学试验提示,与HCP组相比,HCP+CIMT组小鼠在疲劳转棒试验(P<0.01)、上肢悬吊试验(P<0.01)中所停留时间均显著延长;并且小鼠脑内M1区c-Jun、miR-182-5p转录水平也同步升高(P<0.001)。结论转录因子c-Jun主要通过靶向结合miR-182-5p启动子上游-71~-65 bp激活其转录活性。本研究从转录水平阐述c-Jun/miR-182-5p调控是CIMT提高HCP上肢功能的重要分子学基础。Objective To investigate whether the transcription factor c-Jun can directly target miR-182-5p,identify the specific binding sites,and explore whether constraint-induced movement therapy(CIMT)can improve upper limb motor function in hemiplegic cerebral palsy(HCP)mice by promoting c-Jun/miR-182-5p transcription.Methods Lentivirus-mediated c-Jun knockdown and overexpression Neuro-2a cell lines were constructed to establish stable cell models.Western blot was used to verify c-Jun protein expression,while RT-qPCR was employed to measure miR-182-5p expression.Chromatin immunoprecipitation(ChIP)was performed to identify the binding sites of c-Jun on the miR-182-5p promoter region.Subsequently,an HCP mouse model was established and subjected to CIMT intervention for 2 weeks.Behavioral tests were conducted to evaluate the effect of CIMT,and RT-qPCR was used to assess c-Jun and miR-182-5p expression in the left motor cortex(M1)of mice.Results Lentiviral transfection of c-Jun knockdown siRNA(si3,viral titer:5E+8 TU/mL)significantly reduced c-Jun protein expression in Neuro-2a cells(P<0.01),whereas c-Jun overexpression(OE,viral titer:5E+8 TU/mL)markedly increased c-Jun expression(P<0.001).miR-182-5p transcription decreased significantly with reduced c-Jun expression(P<0.01)and increased significantly with elevated c-Jun expression(P≤0.001),demonstrating a synergistic relationship.Further studies revealed that c-Jun binds to multiple sites in the-1,000 to-1 bp upstream region of the miR-182-5p promoter,with the most prominent binding at Site 2(-71 to-65 bp)(P<0.001).Behavioral tests showed that,compared to the HCP group,the HCP+CIMT group exhibited significantly prolonged retention times in the fatigue rotarod test(P<0.01)and the upper limb suspension test(P<0.01).Additionally,c-Jun and miR-182-5p transcription levels in the M1 region of the brain were significantly elevated in the HCP+CIMT group(P<0.001).Conclusion The transcription factor c-Jun enhances miR-182-5p transcription by binding to its upstream promoter region(

关 键 词：C-JUN miR-182-5p 转录调控 偏瘫型脑瘫 强制性运动疗法 

分 类 号：R493[医药卫生—康复医学] R319[医药卫生—临床医学]