miR-17-5p Targeting Autophagy Related Protein ATG7 Regulates Macrophages against <i>Mycobacterium tuberculosis</i> Infection

doi:10.13523/j.cb.20190601

China Biotechnology

2019, Vol. 39

Issue (6): 1-8 DOI: 10.13523/j.cb.20190601

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 XU^1,²,Zheng-hao HUO^1,^4,^**(

),Le GUO^1,^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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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

Received: 21 December 2018 Published: 12 July 2019

ZTFLH:

Q819

Corresponding Authors: Zheng-hao HUO,Le GUO E-mail: huozhh@nxmc.edu.cn;guole@nxmu.edu.cn

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	Dan-tong HONG
	Fan ZHANG
	Shu-e WANG
	Hong-xia WANG
	Kun-mei LIU
	Guang-xian XU
	Zheng-hao HUO
	Le GUO

Cite this article:

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.

URL:

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

Fig.1 Relative expression of miR-17-5p in THP-1 infected by H37Ra with different MOI

Fig.2 Identification of recombinant plasmids (a)Identification of recombinant plasmid pMirGLO-ATG7-3'UTR-WT by double digestion 1: Recombinant plasmid; 2: Plasmid digested by XhoI and SalI; 3:DNA marker (b)Gene sequencing of recombinant plasmid pMirGLO-ATG7-3'UTR-WT (part) (c) pMirGLO-ATG7-3'UTR-MUT by double digestion 1:Recombinant plasmid; 2:Plasmid digested by XhoI and SalI; 3:DNA marker (d)Gene sequencing of recombinant plasmid pMirGLO-ATG7-3'UTR-MUT (part)

Fig.3 Identification of miR-17-5p targeting ATG7 (a)Part of mature miR-17-5p and ATG7 wild type and mutant sequence (b)Transfection efficiency of miR-17-5p mimics and miR-17-5p inhibitor(observed by ordinary light microscope and fluorescence microscope respectively, 10×10) (c)Result of relative expression of miR-17-5p by qRT-PCR (d)Detection of ATG7 relative luciferase activity by dual luciferase report system (e)Results of ATG7 expression by Western blot

Fig.4 Effects of miR-17-5p on autophagy flow and autophagosome formation (a)LC3 II by cell immunofluorescence (b)Results of LC3Ⅱexpression by Western blot


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