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
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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(p Mir GLO-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。
Objective: To explore the role and mechanism of miR-17-5 p in the autophagy pathway mediated by Mycobacterium tuberculosis by studying the regulatory mechanism of miR-17-5 p on autophagy-related gene ATG7 and its effect on cell autophagy. Methods: The target gene ATG7 of miR-17-5 p 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-17-5 p and ATG7 was verified by double luciferase reporting system and Western blot. THP-1-derived macrophages infected by Mycobacterium tuberculosis( H37 Ra) were divided into three groups: miR-17-5 p mimics,miR-17-5 p inhibitors,and miR-17-5 p nc. The effect of H37 Ra infection on the expression of miR-17-5 p was detected by quantitative real-time PCR( qRTPCR). The expression of LC3 protein and the number of autophagosomes were detected by Western blot and immunofluorescence. Results: MTB infection can cause miR-17-5 p down-regulation, with the increase of infection plural decreased significantly. Bioinformatics predictions showed that miR-17-5 p and ATG7 were targeted. Dual luciferase reporter assay and Western blot confirmed that miR-17-5 p 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-17-5 p mimics group,but the reverse was found in the miR-17-5 p inhibitor group. The expression of ATG7 and LC3 II protein in H37 Ra infected group was higher than that in uninfected group. Conclusion: miR-17-5 p directly targets ATG7 3'UTR to inhibit autophagy and plays a role in the anti-MTB effect of macrophages.
Objective: To explore the role and mechanism of miR-17-5 p in the autophagy pathway mediated by Mycobacterium tuberculosis by studying the regulatory mechanism of miR-17-5 p on autophagy-related gene ATG7 and its effect on cell autophagy. Methods: The target gene ATG7 of miR-17-5 p 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-17-5 p and ATG7 was verified by double luciferase reporting system and Western blot. THP-1-derived macrophages infected by Mycobacterium tuberculosis( H37 Ra) were divided into three groups: miR-17-5 p mimics,miR-17-5 p inhibitors,and miR-17-5 p nc. The effect of H37 Ra infection on the expression of miR-17-5 p was detected by quantitative real-time PCR( qRTPCR). The expression of LC3 protein and the number of autophagosomes were detected by Western blot and immunofluorescence. Results: MTB infection can cause miR-17-5 p down-regulation, with the increase of infection plural decreased significantly. Bioinformatics predictions showed that miR-17-5 p and ATG7 were targeted. Dual luciferase reporter assay and Western blot confirmed that miR-17-5 p 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-17-5 p mimics group,but the reverse was found in the miR-17-5 p inhibitor group. The expression of ATG7 and LC3 II protein in H37 Ra infected group was higher than that in uninfected group. Conclusion: miR-17-5 p directly targets ATG7 3'UTR to inhibit autophagy and plays a role in the anti-MTB effect of macrophages.
引文
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[26]Chen Z,Wang T,Liu Z,et al. Inhibition of autophagy by miR-30A induced by mycobacteria tuberculosis as a Possible mechanism of immune escape in human macrophages. Japanese Journal of Infectious Diseases,2015,68(5):420-424.
[27]Panneerdoss S,Viswanadhapalli S,Abdelfattah N,et al. Crosstalk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis(LAP)of apoptotic germ cells. Nature Communications,2017,8(1):598.
[28]Cao W,Qian G,Luo W,et al. MiR-125b is downregulated in systemic lupus erythematosus patients and inhibits autophagy by targeting UVRAG. Biomedicine&Pharmacotherapy=Biomedecine&Pharmacotherapie,2018,99:791-797.
[29]Wang N,Yang L,Zhang H,et al. MicroRNA-9a-5p alleviates ischemia injury after focalcerebral ischemia of the rat by targeting ATG5-Mediated autophagy. Cellular Physiology And Biochemistry:International Journal of Experimental Cellular Physiology,Biochemistry,and Pharmacology,2018,45(1):78-87
[30] Wang X X,Zhang R,Li Y. Expression of the miR-148/152family in acute myeloid leukemia and its clinical significance.Medical Science Monitor:International Medical Journal of Experimental and Clinical Research,2017,23:4768-4778.
[2] World Health Organization. Global tuberculosis report 2018.[2018-12-08]. https://www. aidsdatahub. org/globaltuberculosis-report-2018-who-2018.
[3]Das R,Koo M S,Kim B H,et al. Macrophage migration inhibitory factor(MIF)is a critical mediator of the innate immune response to Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America,2013,110(32):E2997-3006.
[4]Paik S,Kim J K,Chung C,et al. Autophagy:A new strategy for host-directed therapy of tuberculosis. Virulence,2018,10(1):1-12.
[5]Kim K H,Lee M S. Autophagy--a key player in cellular and body metabolism. Nature Reviews Endocrinology,2014,10(6):322-337.
[6]Papackova Z,Cahova M. Important role of autophagy in regulation of metabolic processes in health,disease and aging. Physiological Research,2014,63(4):409-420.
[7]Frudd K,Burgoyne T,Burgoyne J R. Oxidation of atg3 and atg7mediates inhibition of autophagy. Nature Communications,2018,9(1):95.
[8] Siddle K J,Tailleux L,Deschamps M,et al. Bacterial infection drives the expression dynamics of microRNAs and their isomiRs.PLo S Genetics,2015,11(3):e1005064.
[9]Li H,Jin X,Chen B,et al. Autophagy-regulating microRNAs:potential targets for improving radiotherapy. Journal of Cancer Research and Clinical Oncology,2018,144(9):1623-1634.
[10]Zhang G,Liu X,Wang W,et al. Down-regulation of miR-20a-5p triggers cell apoptosis to facilitate mycobacterial clearance through targeting JNK2 in human macrophages. Cell Cycle,2016,15(18):2527-2538.
[11] Yang X,Bai F,Xu Y,et al. Intensified Beclin-1 mediated by low expression of mir-30a-5p promotes chemoresistance in human small cell lung cancer. Cellular Physiology and Biochemistry:International Journal of Experimental Cellular Physiology,Biochemistry,and Pharmacology,2017,43(3):1126-1139.
[12]Li W,Jiang Y,Wang Y,et al. MiR-181b regulates autophagy in a model of Parkinson’s disease by targeting the PTEN/Akt/m TOR signaling pathway. Neuroscience Letters,2018,675:83-88.
[13]Yu K,Li N,Cheng Q,et al. MiR-96-5p prevents hepatic stellate cell activation by inhibiting autophagy via ATG7. Journal of Molecular Medicine,2018,96(1):65-74.
[14] Etna M P,Sinigaglia A,Grassi A,et al. Mycobacterium tuberculosis-induced miR-155 subverts autophagy by targeting ATG3 in human dendritic cells. PLo S Pathogens,2018,14(1):e1006790.
[15] Kim J K,Lee H M,Park K S,et al. Mir144*inhibits antimicrobial responses against mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2. Autophagy,2017,13(2):423-441.
[16] Kim J K,Yuk J M,Kim S Y,et al. MicroRNA-125a inhibits autophagy activation and antimicrobial responses during mycobacterial infection. Journal of Immunology,2015,194(11):5355-5365.
[17] Ouimet M, Koster S, Sakowski E, et al. Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism. Nature Immunology,2016,17(6):677-686.
[18] Ota A, Tagawa H, Karnan S, et al. Identification and characterization of a novel gene,C13orf25,as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Research,2004,64(9):3087-3095.
[19]Yang F,Lei Y,Zhou M,et al. Development and application of a recombination-based library versus library high-throughput yeast two-hybrid(RLL-Y2H)screening system. Nucleic Acids Research,2018,46(3):e17.
[20]Gomez-Sanchez R,Yakhine-Diop S M,Rodriguez-Arribas M,et al. MRAN and protein dataset of autophagy markers(LC3 and p62)in several cell lines. Data In Brief,2016,7:641-647.
[21] Hmama Z,Pena-Diaz S,Joseph S,et al. Immunoevasion and immunosuppression of the macrophage by Mycobacterium tuberculosis. Immunological Reviews,2015,264(1):220-232.
[22] Wan G,Xie W,Liu Z,et al. Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway. Autophagy,2014,10(1):70-79.
[23] Xu G,Zhang Z,Xing Y,et al. MicroRNA-149 negatively regulates TLR-triggered inflammatory response in macrophages by targeting My D88. Journal of Cellular Biochemistry,2014,115(5):919-927.
[24]Zulauf K E,Sullivan J T,Braunstein M. The sec A2 pathway of mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation. PLo S Pathogens,2018,14(4):e1007011.
[25] Sahu S K,Kumar M,Chakraborty S,et al. MicroRNA 26a(miR-26a)/KLF4 and CREB-C/EBPbeta regulate innate immune signaling,the polarization of macrophages and the trafficking of mycobacterium tuberculosis to lysosomes during infection. PLo S Pathogens,2017,13(5):e1006410.
[26]Chen Z,Wang T,Liu Z,et al. Inhibition of autophagy by miR-30A induced by mycobacteria tuberculosis as a Possible mechanism of immune escape in human macrophages. Japanese Journal of Infectious Diseases,2015,68(5):420-424.
[27]Panneerdoss S,Viswanadhapalli S,Abdelfattah N,et al. Crosstalk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis(LAP)of apoptotic germ cells. Nature Communications,2017,8(1):598.
[28]Cao W,Qian G,Luo W,et al. MiR-125b is downregulated in systemic lupus erythematosus patients and inhibits autophagy by targeting UVRAG. Biomedicine&Pharmacotherapy=Biomedecine&Pharmacotherapie,2018,99:791-797.
[29]Wang N,Yang L,Zhang H,et al. MicroRNA-9a-5p alleviates ischemia injury after focalcerebral ischemia of the rat by targeting ATG5-Mediated autophagy. Cellular Physiology And Biochemistry:International Journal of Experimental Cellular Physiology,Biochemistry,and Pharmacology,2018,45(1):78-87
[30] Wang X X,Zhang R,Li Y. Expression of the miR-148/152family in acute myeloid leukemia and its clinical significance.Medical Science Monitor:International Medical Journal of Experimental and Clinical Research,2017,23:4768-4778.