miR-20a-3p靶向STAT3调控大鼠脊髓损伤轴突修复

doi:10.3969/j.issn.1006-9771.2019.03.012

《中国康复理论与实践》 ›› 2019, Vol. 25 ›› Issue (3): 307-313.doi: 10.3969/j.issn.1006-9771.2019.03.012

miR-20a-3p靶向STAT3调控大鼠脊髓损伤轴突修复

张衍军¹, 王志杰², 王天仪³, 陈学明¹, 崔利宾¹, 许崧杰¹, 袁鑫¹, 刘亚东¹, 赵鹏¹, 吴启超¹

1.首都医科大学附属北京潞河医院骨科，北京市 101149
2.承德医学院附属医院儿内科，河北承德市 067000
3.解放军266医院骨一科，河北承德市 067000

收稿日期:2018-09-04 修回日期:2018-12-10 出版日期:2019-03-25 发布日期:2019-04-02
通讯作者: 陈学明,E-mail: xuemingchen@sina.com
作者简介:张衍军(1986-)，男，汉族，山东东平县人，硕士，医师，主要研究方向：脊柱脊髓损伤。
基金资助:
1.首都医科大学附属北京潞河医院公共实验平台培育项目(No. lh201425实)；2.河北省自然科学基金面上项目(No. H2017101030)；3.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(No. 16QNP074)；4.总后卫生部全军医学科技青年培育项目(No. 13QNP017)；5.承德市科学技术研究与发展计划(No. 201606A062; No. 201701A125; No. 201701A127)

Role of MiR-20a-3p in Axonal Repair in Rats with Spinal Cord Injury

ZHANG Yan-jun¹, WANG Zhi-jie², WANG Tian-yi³, CHEN Xue-ming¹, CUI Li-bin¹, XU Song-jie¹, YUAN Xin¹, LIU Ya-dong¹, ZHAO Peng¹, WU Qi-chao¹

1.Department of Orthopedics, Beijing Luhe Hospital, Capital Medical University, Beijing 101149, China
2.Department of Pediatric Internal Medicine, Affiliated Hospital of Chengde Medical College, Chengde, Hebei 067000, China
3.Department of Orthopedics, the 266th Hospital of the Chinese People’s Liberation Army, Chengde, Hebei 067000, China

Received:2018-09-04 Revised:2018-12-10 Published:2019-03-25 Online:2019-04-02
Contact: CHEN Xue-ming, E-mail: xuemingchen@sina.com
Supported by:
Public Experimental Platform Cultivation of Beijing Luhe Hospital, Capital Medical University (No. lh201425), Hebei Natural Science Foundation （General) (No. H2017101030), Central Military Commission Logistics Support Department Health Bureau Military Medicine Science and Technology Youth Cultivation Program Growth Project (No. 16QNP074), Ministry of Health Military Medicine Science and Technology Youth Cultivation Project (No. 13QNP017) and Chengde Science and Technology Research Development Program (No. 201606A062; No. 201701A125; No. 201701A127)

摘要/Abstract

摘要： 目的探索Wistar大鼠脊髓损伤后损伤局部微环境发生改变的分子生物学机制，寻找起关键调控作用的微RNA。方法 15只雌性Wistar大鼠随机分为对照组(n = 3)和脊髓损伤组(n = 12)。脊髓损伤组根据取材时间分为4 h、3 d、7 d、14 d四个组，每组3只。使用Microarray 3.0芯片检测脊髓损伤大鼠损伤局部发生改变的微RNA，运用生物信息学方法论证发挥关键调控作用的微RNA，进行靶基因预测。运用逆转录实时定量聚合酶链反应(RT-qPCR)技术检测miR-20a-3p表达。采用Western blotting技术检测信号传导子和转录激活子(STAT)3表达量，并分析各组中靶蛋白与目标微RNA表达变化趋势相关性。在神经元中抑制关键候选微RNA，并使用免疫荧光观察靶蛋白表达与轴突生长的关系。结果脊髓损伤标本中miR-20a-3p特征性上调明显。生物信息分析结果显示，STAT3可为miR-20a-3p的靶基因，与其趋势相反。细胞实验结果显示，miR-20a-3p抑制组与对照组相比轴突延长。Western blotting结果显示，与对照组相比，miR-20a-3p抑制组STAT3蛋白表达显著上调。结论脊髓损伤后miR-20a-3p通过调节其序列互补靶基因STAT3表达量来影响神经元轴突的生长。miR-20a-3p上调导致STAT3下调，抑制miR-20a-3p，可促进神经元轴突的再生。

关键词: 脊髓损伤, 神经元, 微RNA, 治疗靶点, 大鼠

Abstract: Objective To study the molecular biology mechanisms of Wistar rats after spinal cord injury, and find out key microRNAs. Methods A total of 15 Wistar rats were divided into control group (n = 3) and spinal cord injury group (n = 12). The latter group was divided into four hours, three days, seven days and 14 days subgroups, with three rats in each subgroup. Microarray 3.0 was used to investigate microRNA expression profiles of Wistar rats with spinal cord injury. Bioinformatics was used to predict microRNAs playing key regulatory roles, and to predict target genes. Reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) was applied to detect the expression of miR-20a-3p. Western blotting was employed to detect the signal transducer and activator of transcription (STAT) 3 level. The correlation between target protein and microRNA expression trend in each group was analyzed. The key microRNA was inhibited in the neurons. The relationship between target protein expression and axon growth was observed with immunofluorescence. Results In the rats with spinal cord injury, totally 658 microRNAs had changed at least once. In all the altered microRNAs, miR-20a-3p was upregulated obviously. It predicted that the target gene of miR-20a-3p was STAT3 via application of bioinformatics analysis. The expression trend of STAT 3 and miR-20a-3p in spinal cord was opposite. After the inhibition of miR-20a-3p, the expression of STAT3 in neurons was unregulated and axonal growth was extended. Conclusion The upregulation of miR-20a-3p leads to downregulation of STAT3, and miR-20a-3p is one of the key targets in the treatment of spinal cord injury.

Key words: spinal cord injury, neuron, microRNA, therapeutic target, rats

中图分类号:

R651.2

张衍军, 王志杰, 王天仪, 陈学明, 崔利宾, 许崧杰, 袁鑫, 刘亚东, 赵鹏, 吴启超. miR-20a-3p靶向STAT3调控大鼠脊髓损伤轴突修复[J]. 《中国康复理论与实践》, 2019, 25(3): 307-313.

ZHANG Yan-jun, WANG Zhi-jie, WANG Tian-yi, CHEN Xue-ming, CUI Li-bin, XU Song-jie, YUAN Xin, LIU Ya-dong, ZHAO Peng, WU Qi-chao. Role of MiR-20a-3p in Axonal Repair in Rats with Spinal Cord Injury[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2019, 25(3): 307-313.

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miR-20a-3p靶向STAT3调控大鼠脊髓损伤轴突修复

Role of MiR-20a-3p in Axonal Repair in Rats with Spinal Cord Injury

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