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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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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.
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Received: 21 December 2018
Published: 12 July 2019
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Corresponding Authors:
Zheng-hao HUO,Le GUO
E-mail: huozhh@nxmc.edu.cn;guole@nxmu.edu.cn
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