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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (6): 1-8    DOI: 10.13523/j.cb.20190601
研究报告     
miR-17-5p靶向自噬相关蛋白ATG7调控巨噬细胞抗结核分枝杆菌感染作用的研究 *
洪丹彤1,张帆1,王淑娥1,王红霞1,刘昆梅3,徐广贤1,2,霍正浩1,4,**(),郭乐1,2,**()
1 宁夏医科大学临床医学院 银川 7500212
2 宁夏医科大学总医院 宁夏临床病原微生物重点实验室 银川 750021
3 宁夏医科大学颅脑疾病重点实验室 银川 750021
4 宁夏医科大学基础医学院 宁夏生殖与遗传重点实验室 银川 750021
miR-17-5p Targeting Autophagy Related Protein ATG7 Regulates Macrophages against Mycobacterium tuberculosis Infection
Dan-tong HONG1,Fan ZHANG1,Shu-e WANG1,Hong-xia WANG1,Kun-mei LIU3,Guang-xian XU1,2,Zheng-hao HUO1,4,**(),Le GUO1,2,**()
1 School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China
2 Ningxia Key Laboratory of Clinical and Pathogenic Microbiology, General Hospital of Ningxia Medical University, Yinchuan 750004, China
3 Ningxia Key Laboratory of Cerebrocranial Diseases, Ningxia Medical University, Yinchuan 750004, China
4 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan 750004, China
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摘要：

目的: 通过研究miR-17-5p对自噬相关基因ATG7的靶向调控机制和对细胞自噬的作用,探究miR-17-5p在结核分枝杆菌介导的自噬途径中的作用及其机制。方法: 生物信息学分析得到miR17-5p的靶基因ATG7,通过成功构建载体ATG7野生型(pMirGLO-ATG7-3'UTR-WT)和突变型,利用双萤光素酶报告系统、Western blot验证miR-17-5p和ATG7的靶向关系,同时构建结核分枝杆菌(H37Ra)感染的人源性THP-1巨噬细胞模型,将做不同处理的细胞分为三组:miR-17-5p mimics、miR-17-5p inhibitor、miR-17-5p nc。通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测H37Ra感染对miR-17-5p表达量的影响,并且进一步通过Western blot、免疫荧光观察检测LC3蛋白的表达量和自噬小体的数量。结果: MTB感染能够引起miR-17-5p的下调,随着感染复数的增加有明显的降低。而生物信息学预测结果显示miR-17-5p与ATG7具有靶向性,双萤光素酶报告实验、Western blot验证miR-17-5p能够和ATG7靶向结合,并对其进行负调控。进一步通过Western blot、免疫荧光观察发现miR-17-5p mimics组LC3Ⅱ的表达下调,自噬小体表达降低,而miR-17-5p inhibitor组相反。其中对H37Ra感染组与未感染组之间比较,ATG7和LC3Ⅱ蛋白表达明显增强。结论: miR-17-5p直接靶向结合ATG7 3'UTR抑制自噬,在巨噬细胞抗MTB过程中发挥作用。
关键词： 自噬;  结核分支杆菌;  miR-17-5p;  ATG7    
Abstract:

Objective:To explore the role and mechanism of miR-17-5p in the autophagy pathway mediated by Mycobacterium tuberculosis by studying the regulatory mechanism of miR-17-5p on autophagy-related gene ATG7 and its effect on cell autophagy. Methods: The target gene ATG7 of miR-17-5p was obtained by bioinformatics analysis. The wild-type(pMirGLO-ATG7-3'UTR-WT) and mutant vector of ATG7 were successfully constructed. The targeting relationship between miR-17-5p and ATG7 was verified by double luciferase reporting system and Western blot. THP-1-derived macrophages infected by Mycobacterium tuberculosis (H37Ra) were divided into three groups: miR-17-5p mimics, miR-17-5p inhibitors, and miR-17-5p nc. The effect of H37Ra infection on the expression of miR-17-5p was detected by quantitative real-time PCR (qRT-PCR). The expression of LC3 protein and the number of autophagosomes were detected by Western blot and immunofluorescence. Results: MTB infection can cause miR-17-5p down-regulation, with the increase of infection plural decreased significantly. Bioinformatics predictions showed that miR-17-5p and ATG7 were targeted. Dual luciferase reporter assay and Western blot confirmed that miR-17-5p could bind to ATG7 and negatively regulate it. Western blot and immunofluorescence assay showed that the expression of LC3 II was down-regulated and the expression of autophagosomes was down-regulated in the miR-17-5p mimics group, but the reverse was found in the miR-17-5p inhibitor group. The expression of ATG7 and LC3 II protein in H37Ra infected group was higher than that in uninfected group. Conclusion: miR-17-5p directly targets ATG7 3'UTR to inhibit autophagy and plays a role in the anti-MTB effect of macrophages.
Key words: Autophagy    Mycobacterium tuberculosis    miR-17-5p    ATG7
收稿日期: 2018-12-21 出版日期: 2019-07-12
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(81760359);国家自然科学基金(31600744);宁夏高等学校科学研究项目资助项目(NGY2017088)
通讯作者: 霍正浩,郭乐     E-mail: huozhh@nxmc.edu.cn;guole@nxmu.edu.cn
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洪丹彤
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刘昆梅
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霍正浩
郭乐

引用本文:

洪丹彤,张帆,王淑娥,王红霞,刘昆梅,徐广贤,霍正浩,郭乐. miR-17-5p靶向自噬相关蛋白ATG7调控巨噬细胞抗结核分枝杆菌感染作用的研究 *[J]. 中国生物工程杂志, 2019, 39(6): 1-8.

Dan-tong HONG,Fan ZHANG,Shu-e WANG,Hong-xia WANG,Kun-mei LIU,Guang-xian XU,Zheng-hao HUO,Le GUO. miR-17-5p Targeting Autophagy Related Protein ATG7 Regulates Macrophages against Mycobacterium tuberculosis Infection. China Biotechnology, 2019, 39(6): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190601    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I6/1

图1  H37Ra以不同感染复数(MOI)处理THP-1细胞后miR-17-5p的表达水平
图2  萤光素酶报告基因重组质粒的鉴定
图3  miR-17-5p对ATG7靶向性的鉴定
图4  miR-17-5p对自噬流及自噬小体形成的作用
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