Figure 4.
Figure 4. miR-125b promotes M1 macrophage polarization. (A) Total RNA was isolated from THP-1 transfected with control miRNA, miR-125b mimics (miR-125b), or miR-125b antagomir (Anti-125b). After reverse transcription, analysis of the expression level of 96 inflammatory genes was performed using a TaqMan-based Low Density Array Human Inflammation Panel. The volcano plot (top) displays statistical significance vs fold-change (FC) pre-miR-125b/antagomiR-125b on the y- and x-axes, respectively. Red dots represent M1 specific genes: TNF, NOS2A, CXCL10, and CXCL11. Graphs (bottom) represent RNA expression levels of these genes as mean ± SD of 3 technical experiments. (B) THP-1 monocytes (MO) were differentiated into macrophages (Mϕ) after 3 days of culture with PMA (0.1 μg/mL), and then polarized into M1 macrophages using IFN-γ and LPS stimulation for 24 hours. M1 polarization phenotype was monitored by quantification of CXCL11 mRNA expression using quantitative reverse transcription polymerase chain reaction (left), and of human TNF (hTNF) secretion using enzyme-linked immunosorbent assay (right). (C) Real-time analysis of the OCR for THP-1 monocytes differentiated into macrophages, polarized or not (open circles) toward M1 phenotype (filled circles). (D) Expression levels of miR-125b, BIK, and MTP18 mRNA in monocytes (MO), macrophages (Mϕ), or M1-polarized macrophages (M1) using real-time polymerase chain reaction. Error bars represent the mean ± SD of 3 independent experiments. (E) Unpolarized (Mϕ) or M1-polarized macrophages (M1) were fixed and stained with anti-TOM20 antibodies. Representative images (left; bar represents 10 μm) and quantification of captured images (right) are shown. (F-G) Expression levels of CXCL11 and TNF mRNAs (F) and human TNF secretion (G) in THP-1 cells transfected with control miRNA (CTRL), miR-125b mimics, or miR-125b antagomir. RNA quantification is presented as fold change to the controls, and error bars represent the SD of 4 independent experiments. *P < .05; **P < .01; ***P < .001. TFN, tumor necrosis factor.

miR-125b promotes M1 macrophage polarization. (A) Total RNA was isolated from THP-1 transfected with control miRNA, miR-125b mimics (miR-125b), or miR-125b antagomir (Anti-125b). After reverse transcription, analysis of the expression level of 96 inflammatory genes was performed using a TaqMan-based Low Density Array Human Inflammation Panel. The volcano plot (top) displays statistical significance vs fold-change (FC) pre-miR-125b/antagomiR-125b on the y- and x-axes, respectively. Red dots represent M1 specific genes: TNF, NOS2A, CXCL10, and CXCL11. Graphs (bottom) represent RNA expression levels of these genes as mean ± SD of 3 technical experiments. (B) THP-1 monocytes (MO) were differentiated into macrophages (Mϕ) after 3 days of culture with PMA (0.1 μg/mL), and then polarized into M1 macrophages using IFN-γ and LPS stimulation for 24 hours. M1 polarization phenotype was monitored by quantification of CXCL11 mRNA expression using quantitative reverse transcription polymerase chain reaction (left), and of human TNF (hTNF) secretion using enzyme-linked immunosorbent assay (right). (C) Real-time analysis of the OCR for THP-1 monocytes differentiated into macrophages, polarized or not (open circles) toward M1 phenotype (filled circles). (D) Expression levels of miR-125b, BIK, and MTP18 mRNA in monocytes (MO), macrophages (Mϕ), or M1-polarized macrophages (M1) using real-time polymerase chain reaction. Error bars represent the mean ± SD of 3 independent experiments. (E) Unpolarized (Mϕ) or M1-polarized macrophages (M1) were fixed and stained with anti-TOM20 antibodies. Representative images (left; bar represents 10 μm) and quantification of captured images (right) are shown. (F-G) Expression levels of CXCL11 and TNF mRNAs (F) and human TNF secretion (G) in THP-1 cells transfected with control miRNA (CTRL), miR-125b mimics, or miR-125b antagomir. RNA quantification is presented as fold change to the controls, and error bars represent the SD of 4 independent experiments. *P < .05; **P < .01; ***P < .001. TFN, tumor necrosis factor.

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