微小RNA-92b在儿童急性横贯性脊髓炎发生机制中的作用  

Role of microRNA-92b in the pathophysiology of acute transverse myelitis in children

作　　者：王志杰[1] 王天仪[2] 刘丽娜[1] 虞妹 李波[5] 刘霞[1] 张铮 周搏 王新 郭晓玲 Wang Zhijie;Wang Tianyi;Liu Lina;Yu Mei;Li Bo;Liu Xia;Zhang Zheng;Zhou Bo;Wang Xin;Guo Xiaoling(Department of Pediatric Internal Medicine,Affiliated Hospital of Chengde Medical College,Chengde 067000,Hebei Provinee,China;Department of Orthopedics,the 266^th Hospital of the Chinese People's Liberation Army,Chengde 067000,Hebei Province,China;Department of Neurology,the 266^th Hospital of the Chinese People's Liberation Army,Chengde 067000,Hebei Province,China;Leukemia Center,Chinese Academy of Medical Sciences & Peking Union of Medical College,Institute of Hematology & Hospital of Blood Diseases,Tianjin 300020,China;Department of Orthopedics,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]承德医学院附属医院儿内科,067000 [2]中国人民解放军第二六六医院骨一科,河北承德067000 [3]中国人民解放军第二六六医院神经内科,河北承德067000 [4]中国医学科学院血液病医院白血病诊疗中心,天津300020 [5]天津医科大学总医院骨科,300052

出　　处：《中华实用儿科临床杂志》2018年第24期1859-1863,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金重点项目(81330042);河北省自然科学基金面上项目(H2017101030);中央军委后勤保障部卫生局全军医学科技青年培育计划成长项目(16QNP074);总后卫生部全军医学科技青年培育项目(13QNP017);承德市科学技术研究与发展计划(201606A062,201701A125,201701A127).

摘　　要：目的筛选调控儿童急性横贯性脊髓炎的关键微小RNA(miRNA),并验证其调控的相关通路。方法本研究纳入12例急性横贯性脊髓炎患者作为ATM组,纳入3例正常脑脊液儿童作为对照组,使用microarray4.0芯片检测儿童急性横贯性脊髓炎患者脑脊液中发生改变的miRNA。运用生物信息学方法论证发挥关键调控作用的miRNA并进行靶基因预测。实时荧光定量PCR(qPCR)技术进行生物学与技术重复。用酶联免疫吸附法(ELISA)与Westernblot技术检测关键miRNA靶蛋白表达量。在背根神经节神经元中抑制/过表达关键候选miRNA,进行体外功能验证。应用夫拉平度(Flavopiridol)抑制CDKs的活性来验证miR-92b是通过p57-CDKs-GAP-43通路发挥作用的。结果急性横贯性脊髓炎患者脑脊液样本中miR-92b特征性上调明显。生物信息分析结果显示p57可为miR-92b的靶基因。miR-92b与p57蛋白样本间表达趋势相反。体外细胞实验显示miR-92b过表达组的神经元轴突生长明显弱于对照组;miR-92b抑制组的神经元轴突明显延长;在miR-92b过表达+Flavopiridol组,背根神经节神经元轴突与对照组相比依旧显著延长。Westernblot实验结果显示miR-92b过表达组的p57和GAP-43蛋白表达低于对照组;与对照组相比,miR-92b抑制组的p57和GAP-43蛋白表达显著上调;而在miR-92b过表达+Flavopiridol组,p57表达低于对照组,GAP-43蛋白的表达量高于对照组。结论儿童急性横贯性脊髓炎患者miR-92b的上调导致了p57的下调,使CDKs的活性增强,进而抑制GAP-43蛋白的表达和脊髓损伤区域轴突的再生。miR-92b是儿童急性横贯性脊髓炎治疗的关键靶点之一。Objective To explore microRNAs that play key regulatory roles in the pathophysiology of acute transverse myelitis in children, and to find therapeutic targets. Methods Twelve patients with acute transverse myelitis were enrolled as ATM group and three children with normal cerebrospinal fluid as the control group.MicroRNA in cerebrospinal fluid of children with acute transverse myelitis was detected by using microarray4.0 chip.Bioinformatics was used to demonstrate microRNA, which plays a key regulatory role, and to predict target genes.Real-time quantitative polymerase chain reaction (qPCR) technique was adopted for in biology and technology duplication.Enzyme-linked immunosorbent assay (ELISA) and Western blot technique were used to detect the expression of key miRNA target protein. The key candidate microRNA was inhibited/overexpressed in dorsal root ganglion neurons, and the function was verified in vitro.Flavopiridol was used to inhibit the activity of CDKs to verify that miR-92b worked through p57-CDKs-GAP-43 pathway. Results The characteristic elevation of miR-92b in cerebrospinal fluid samples of acute transverse myelitis was significant. Bioinformatics analysis showed that p57 was the target gene of miR-92b.The expression of miR-92b was contrary to the p57 protein.In vitro experiments showed that the length of axons in miR-92b mimics group was significantly shorter than that in the blank group.The axons of neurons in antimiR-92b group were obviously prolonged. In the miR-92b mimics+ Flavopiridol groups, the axons of neurons were still significantly prolonged compared with that in the blank group.Western blot showed that p57 and GAP-43 protein expression in miR-92b mimics group was lower than that in blank group.The expression of p57 and GAP-43 protein in antimiR-92b group was significantly higher than that in blank group.But in miR-92b mimics+ Flavopiridol group, the expression of p57 was lower compared with that in blank group, and the expression of GAP-43 protein was higher compared with that in blank g

关 键 词：急性横贯性脊髓炎 儿童 脑脊液 微小RNA 治疗靶点 

分 类 号：R744.3[医药卫生—神经病学与精神病学]