Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: Sequences of the Primers Used for qRT-PCR

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Sequencing

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: IRF1 is a target gene of miR-106b-5p in the glioma infiltrating macrophages. ( A ) The predicted miR-106b-5p-binding site of the 3ʹ-UTR, and mutation of IRF1 3ʹ-UTR disrupted miR-106b-5p binding. ( B ) Luciferase activity assay showed the binding of miR-106b-5p to the 3ʹUTR of IRF1 and inhibition of IRF1 (** P <0.01 vs scramble). ( C ) IRF1 expression was significantly upregulated in M1 subset and downregulated in M2 subset in THP-1 and Raw264.7 cells (* P <0.05, ** P <0.01 vs M0). ( D ) IRF1 expression was significantly upregulated in M1 macrophages and downregulated in M2 macrophages in murine peritoneal macrophages (* P <0.05 vs M0). ( E ) Western blotting of IRF1 in M1, M2 and M0 macrophages (* P <0.05). ( F ) IRF1 expression was detected after transfection with miR-106b-5p mimics or inhibitor in THP1 and Raw264.7 cells. RF1 expression decreased after miR-106b-5p mimics treatment, and increased after miR-106b inhibitor treatment (* P <0.05, ** P <0.01 vs NC). ( G ) Protein expression of IRF1 after transfection with miR-106b-5p mimics or inhibitor; * P <0.05.

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Inhibition, Expressing, Western Blot, Transfection

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: miR-106b-5p expression in the glioblastoma and syngeneic intracranial glioma model. ( A ) In the in situ hybridization, digoxigenin-conjugated oligonucleotide miR-106b-5p probe was used to detect miR-106b-5p expression in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues (n=3, 100×). ( B ) Immunohistochemistry for Ki67 in the glioblastoma. Left: normal brain tissues. Right: glioblastoma tissues. ( C ) In the in situ hybridization-, digoxigenin-conjugated oligonucleotide miR-106b-5p probe to detect miR-106b-5p expression in the syngeneic intracranial glioma models (n=3, 100×). ( D ) Immunohistochemistry for Ki67 in the syngeneic intracranial glioma models (n=3, 100×). ( E ) IRF1 expression in the syngeneic intracranial glioma models (qRT-PCR, *** P <0.001). ( F ) IRF1 expression in the syngeneic intracranial glioma models (Western blotting; n=3).

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: miR-106b-5p enhanced the proliferation of glioma-infiltrating macrophages to increase tumor growth. ( A ) The time points of tumor cell inoculation and measurements; ( B, C ) volumes of subcutaneous GL261 tumor in the miR-106b-5p mimics/inhibitor group and NC group (* P <0.05 vs control, n=10). ( D ) Subcutaneous GL261 tumor in C57BL/6J mice. ( E ) IRF1 protein expression (IHC; 100×, n=3). ( F ) Ki67 protein expression (IHC; 100×, n=3). ( G ) CD163 protein expression (IHC; 100×, n=3).

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Control, Expressing

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: miR-106b-5p promoted M2 polarization of macrophages by targeting IRF1/IFN-β pathway. ( A, B ) Over-expression/silencing of IRF1 could reverse the down-regulated and up-regulated IRF1 expression by miR-106b-5p mimics and inhibitor, respectively (* P <0.05, ** P <0.01 vs control). ( C ) The mRNA expressions of IFN-β and IRF5 significantly decreased in THP-1 cells treated with conditioned medium from U251 cell as glioma-infiltrating macrophage model (* P <0.05, ** P <0.01 vs control). ( D ) The mRNA expressions of IFN-β and IRF5 significantly increased in M1 macrophages, and decreased in M2 macrophage (* P <0.05, ** P <0.01 vs M0). ( E ) The mRNA expressions of IFN-β and IRF5 increased after overexpression of IRF1 or inhibition of miR-106b-5p (* P <0.05, ** P <0.01 vs control). ( F ) The mRNA expression of IRF5, IL-10 and CD163 in THP1 and Raw264.7 cells transfected with IRF5 or siIRF5 and miR-106b-5p mimics or inhibitor. ( G ) The mRNA expression of IFN-β, IL-10 and CD163 in THP1 and Raw264.7 cells transfected with IFN-β or siIFN-β and miR-106b-5p mimics or inhibitor.

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Over Expression, Expressing, Control, Inhibition, Transfection

Journal: OncoTargets and therapy

Article Title: miR-106b-5p Inhibits IRF1/IFN-β Signaling to Promote M2 Macrophage Polarization of Glioblastoma

doi: 10.2147/OTT.S238975

Figure Lengend Snippet: IRF1 regulates miR-106b-5p in M2 macrophage polarization. Our findings suggest, in glioma tumor microenvironment, miR-106b-5p expression is down-regulated in M1 macrophages, but up-regulated in M2 macrophages. miR-106b-5p binds to IRF1 to inhibit IRF1 expression in glioma tumor microenvironment. Macrophages are plastic cell population, and undergo a phenotypically dynamic switch between M1 and M2 macrophages. IRF1, IFN-β and IRF5 interact with each other to promote M1 polarization. We speculate that the decrease of IRF1 may block the interaction of IRF1/IFN-β/IRF5 and promote M1 to M2 polarization. This is important for the glioma tumor growth.

Article Snippet: Then, sections were incubated with primary antibody overnight at 4°C (IRF1 [#8478, Cell Signaling Technology], CD163 [ab182422, Abcam] and Ki67 [ab15580, Abcam]), followed by incubation with horse radish peroxidase (HRP) conjugated secondary antibody for 30 min at room temperature.

Techniques: Expressing, Blocking Assay

Journal: Journal of neuroinflammation

Article Title: GPR34 in spinal microglia exacerbates neuropathic pain in mice.

doi: 10.1186/s12974-019-1458-8

Figure Lengend Snippet: Fig. 1 GPR34 expression is induced in microglia in the spinal dorsal horn after L4 nerve injury. a qRT-PCR analysis of GPR34 mRNA in the ipsilateral L4 dorsal horn of WT mice before (Naive) and after L4 nerve injury. Results are normalized to the housekeeping gene GAPDH (n = 4 for each time point). Values are mean ± SEM. Data are shown as fold change over naive sample. *p < 0.05, **p < 0.01 (one-way ANOVA with post hoc Tukey’s test). b, c In situ hybridization shows increased GPR34 mRNA expression in the spinal dorsal horn 7 days after L4 nerve injury (low-magnification images). Contra contralateral side, Ipsi ipsilateral side. Scale bar = 200 μm. d–f Localization of GPR34 mRNA in microglia in the ipsilateral dorsal horn (high-magnification images). Hybridized cells (d) display IRF8 immunoreactivity (e) in their nuclei (arrows). Scale bar = 30 μm

Article Snippet: Primary antibodies were as follows: anti-ionized calcium-binding adaptor molecule 1 (Iba1) (Abcam, RRID: AB_2224402; 1:500), anti-interferon regulatory factor 8 (IRF8) (Santa Cruz Biotechnology, RRID: AB_649510; 1:250), and anti-protein kinase C gamma (PKCγ) (#sc-211, RRID: AB_632234; Santa Cruz Biotechnology; 1:500).

Techniques: Expressing, Quantitative RT-PCR, In Situ Hybridization