Fig 1.
miR-144-3p expression is induced after BCG infection.
(A) RAW264.7 cells were infected with BCG at different MOI for 24 h, and the expression levels of miR-144-3p were measured by qRT-PCR. The normalized miR-144-3p expression for control (MOI = 0) was set as 1. Data are shown as the mean ± SD of at least three independent experiments. **, p<0.01; ***, p <0.001. (B) miR-144-3p expression levels were examined after infected with BCG at an MOI of 10 for the indicated times, and the expression levels of miR-144-3p were measured by qRT-PCR. The normalized miR-144-3p expression for control (the infection time = 0) was set as 1. Data are shown as the mean ± SD of at least three independent experiments. *, p <0.05; **, p<0.01; ***, p <0.001.
Fig 2.
miR-144-3p represses ATG4a expression by targeting its 3' UTR.
(A) Bioinformatic analysis was used to predict the mouse miR-144-3p seed sequences and the 3'UTR sequences of wild type (WT) and mutant ATG4a. (B) The expression level of miR-144-3p in RAW264.7 cells transfected with miR-144-3p control, miR-144-3p mimic or its inhibitor was determined by a qRT-PCR assay. qRT-PCR data were normalized with U6 small nucleolus RNA data. Data represent the mean ± SD from three independent experiments. **p<0.01. (C) RAW264.7 cells were cotransfected with pMIR-REPORT Luciferase vector carrying wild-type ATG4a or mutant ATG4a, along with miR-144-3p control or miR-144-3p mimics, and a luciferase assay was performed. Data are shown as mean ± SD of four independent experiments. **p<0.01. (D) RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor. 50 μg of total cell extracts were analyzed by Western blotting with a rabbit anti-ATG4a antibody. Representative Western blot image for ATG4a expression and quantitative analysis of ATG4a protein levels, normalized to GAPDH expression, are shown. Data are shown as mean ± SD of four independent experiments. *, p <0.05; **, p<0.01. (E) RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor. The expression levels of ATG4a mRNA were detected by qRT-PCR. Data represent the mean ± SD from three independent experiments. **p<0.01, ***p<0.001.
Fig 3.
miR-144-3p inhibits autophagy in RAW264.7 cells.
(A and B) RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or inhibitor, and then infected with BCG for 24 h. The cells were incubated with a rabbit anti-LC3 Ab, followed by detection using Alexa Fluor 488–conjugated goat anti-rabbit IgG and DAPI. The formation of LC3 puncta was then detected by confocal microscopy. Scale bar = 5 μm. The control cells are BCG-infected cells without transfection with any types of miRNAs. LC3 puncta were determined using ImageJ software (miR-144-3p control, n = 15 cells; miR-144-3p mimic, n = 15 cells; miR-144-3p inhibitor, n = 15 cells). Data represent the means ± SD from three independent experiments. *p < 0.05, **p < 0.01. (C) RAW264.7 cells were transfected with an miR-144-3p mimic or inhibitor and then infected with BCG for 24 h. 30 μg of total cell extracts were analyzed by Western blotting with a rabbit anti-LC3 antibody. The conversion of LC3-I to LC3-II was determined by Western blot analysis (upper). The quantitative analysis of LC3-II protein levels, normalized to β-actin expression, are shown (lower). Data are shown as mean ± SD of three independent experiments. *, p <0.05; **, p<0.01. (D) RAW264.7 cells transfected with miR-144-3p control or miR-144-3p mimic in full medium with or without 50μg/ml rapamycin or 0.1% DMSO. 50 μg of total cell extracts were analyzed by Western blotting with a mouse anti-SQSTM1/p62 antibody. The p62 levels were detected by Western-blot (upper). Quantitative analysis of the p62 band normalized to β-actin is shown (lower). Data are shown as mean ± SD of four independent experiments. *, p <0.05. (E) RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or inhibitor and then infected with BCG. 50 μg of total cell extracts were analyzed by Western blotting with a rabbit anti-ATG4a antibody. Representative Western blot image for ATG4a expression (upper) and quantitative analysis of ATG4a protein levels, normalized to β-actin expression (lower), are shown. Data are shown as mean ± SD of three independent experiments. *, p <0.05; **, p<0.01. (F) miR-144-3p inhibited autolysosome formation. RAW264.7 cells were transiently transfected with miR-144-3p control, miR-144-3p mimic or inhibitor, or were treated with rapamycin, chloroquine, or rapamycin plus miR-144-3p mimic. Then RAW264.7 cells were infected with BCG for 24 h. After that the RAW264.7 cells were incubated with a specific fluorescent dye Lyso tracker RED (50 nM). The control cells are BCG-infected cells without transfection with any types of miRNAs. The positive acid compartments were detected by confocal microscopy. Scale bar = 5 μm.
Fig 4.
miR-144-3p inhibited autophagosome formation.
(A and B) Transmission electron microscopy confirms repression of autophagy by miR-144-3p overexpression or knockdown. RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor for 24 h, and then infected with BCG for 24 h. M, mitochondrion. Autophagosomes (denoted by red arrows). The number of autophagosomes per cross-sectioned cell was counted (15 cells per group counted by TEM). *p<0.05, **p<0.01. (C and D) Overexpression of miR-144-3p could attenuate induction of autophagy by rapamycin. The BCG-challenged RAW264.7 cells were transfected with miR-144-3p mimics, and then treated with 50 μg/ml rapamycin for 2 h. Meanwhile the BCG-challenged RAW264.7 cells were treated with rapamycin or 0.1% DMSO as controls. BCG bacteria (denoted by black arrows). M, mitochondrion. Autophagosomes (denoted by red arrows). The number of autophagosomes per cross-sectioned cell was counted (15 cells per group counted by TEM). **p<0.01, ***p<0.001.
Fig 5.
Intracellular BCG survival analyzed by qPCR and CFU assay.
(A) The RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or inhibitor, or were treated with rapamycin or 3-MA, and then infected with BCG for 24 h. The DNA of intracellular BCG was determined by using a qPCR assay. Cells trated with rapamycin or 3-MA were used as a positive or negtive control of autophagy. *p<0.05, **p<0.01, ***p<0.001. (B) The RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor, or were treated with rapamycin or 3-MA. After that, the RAW264.7 cells were infected with BCG for 24 h, 48 h and 72 h. Intracellular BCG survival was evaluated by CFU assay. *p<0.05, **p<0.01, ***p<0.001.
Fig 6.
Schematic representation of miR-144-3p inhibiting autophagy activation during mycobacterial infection by targeting ATG4a.
MiR-144-3p overexpression in macrophages directly represses ATG4a by binding to its 3’UTR, which in turn inhibits the formation of autophagosomes in macrophages and increases the survival rate of intracellular mycobacteria.