Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: (A and B) Monocytes isolated from BM of WT or miR-148a–/– mice were cultured with 50 ng/mL GM-CSF and 20 ng/mL IL-4 for 5 days. Flow cytometry analysis (A), morphology (B), and statistical data (C) of moDCs and macrophages are shown. (D) Flow cytometry and statistical data of apoptosis during monocyte differentiation at indicated time points were analyzed (n = 3). Scale bars: 10 μm (B). P values were determined by 2-tailed unpaired t test. Data are shown as mean ± SEM. (***P < 0.001, n.s., not significant). miR-148a, microRNA-148a; moDC, monocyte-derived DC.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Isolation, Cell Culture, Flow Cytometry, Derivative Assay

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: (A and B) Monocytes isolated from miR-148a–/– or WT BM were cultured with GM-CSF and IL-4, or not. Gene expression were detected by mRNA sequencing. (A) Gene Ontology analysis of differentially expressed genes. (B) Venn diagram of upregulated genes in miR-148a–/– monocytes on days 0 and 3 is shown. Upregulated genes with predicted miR-148a targets are listed (n = 3). (C) Predicted binding site of miR-148a in the 3′UTR of MAFB and the mutation of binding site. (D) Immunoblot analysis of MAFB expression in monocytes isolated from WT or miR-148a–/– BM. The target gene protein/β-ACTIN ratio in the WT group was set as 1. (E) RNA immunopurification assay. Human primary monocytes were transfected with control (NC) or Agomir-148a. QRT-PCR analysis of enriched MAFB mRNA levels after Ago2 immunoprecipitation. IgG was used as negative control (n = 3). (F) Dual-luciferase reporter assays showing direct interaction of miR-148a with its cognate binding sites in the 3′UTR of MAFB. MAFB 3′UTR mutant refers to construct with mutated cognate binding sites for miR-148a. The renilla luciferase (Renilla) signal regulated by the target genes 3′UTR was normalized by the firefly luciferase (Firefly). The Renilla/Firefly ratio was set as 1 for the control psiCHECK2 plasmid (n = 3). (G) Knockdown of MAFB reduced the difference in the differentiation of moDCs between WT and miR-148a–/– monocytes. Monocytes derived from WT and miR-148a–/– mice were transfected with control (NC) or MAFB siRNA and then cultured with GM-CSF and IL-4 for indicated days. The differentiation of moDCs was analyzed (n = 3). Statistical analysis was performed with 2-tailed unpaired t test or 1-way ANOVA with Bonferroni’s post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Isolation, Cell Culture, Gene Expression, Sequencing, Binding Assay, Mutagenesis, Western Blot, Expressing, Immu-Puri, Transfection, Control, Quantitative RT-PCR, Immunoprecipitation, Negative Control, Luciferase, Construct, Plasmid Preparation, Knockdown, Derivative Assay

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: (A) Sequence alignment of the human and mouse miR-148a promoter (-strand) showing localization of predicted PU.1 binding site (blue). The transcriptional start sites are highlighted in red. Genomic locations for the human (GRCh38/hg18) and mouse (GRCm38/mm10) sequence intervals are provided above. (B) TaqMan qRT-PCR analysis of miR-148a expression in control (NC) or PU.1 siRNA transfected human monocytes. U6 snRNA was used as endogenous control (n = 3). (C and D) Kinetic analysis of miR-148a, PU.1 and MAFB expression during moDC differentiation. (C) mRNA levels of PU.1 and MAFB were detected by qRT-PCR and normalized to the β-ACTIN RNA level. The mRNA/β-ACTIN ratio on day 0 was set as 1. (D) Immunoblot analysis. The protein/β-ACTIN ratio on day 0 was set as 1. (E) Monocytes isolated from WT and miR-148a–/– BM cells were transfected with control (NC) or siRNA specific to PU.1 (si-PU.1), and the protein levels of MAFB were detected. The MAFB/β-ACTIN ratio in WT+NC was set as 1. (F) Luciferase reporter assay in Hela cells cotransfected with the promoter of miR-148a (148-luc) or miR-148a promoter lacking the PU.1 binding site (mut-luc), and a vector overexpressing PU.1. The firefly luciferase signal was normalized by renilla luciferase. The Firefly/Renilla ratio was set as 1 for the no promoter empty plasmid (luc) (n = 3). (G) Binding of PU.1 within the miR-148a promoter was analyzed using ChIP and qRT-PCR on human monocytes collected on day 0 or day 3 during moDC differentiation. The ets site in the CD11b promoter was set as positive control, whereas the downstream of miR-148 TSS was set as negative control (neg). Data are the mean percentage of input ± SEM (n = 3). P values compare the indicated groups using a 2-tailed unpaired t test or 1-way ANOVA with Bonferroni’s post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a; moDC, monocyte-derived DC; quantitative real-time PCR, qRT-PCR.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Control, Transfection, Western Blot, Isolation, Luciferase, Reporter Assay, Plasmid Preparation, Positive Control, Negative Control, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: WT and miR-148a–/– mice were treated with IMQ for 6 consecutive days. (A) Phenotypic presentation of skin lesions of representative mice on day 6. (B) Ear thickness. (C) PASI score. (D) H&E staining. (E) Dermal thickness. (F) Acanthosis. (n = 5). (G) The relative mRNA expression of indicated cytokines in skin (top) and the protein levels (bottom) of these cytokines in serum on day 4 (n = 5). (H) Flow cytometry analysis of the skin infiltration of moDCs (CD45+MHCIIhiLy6C–CD64–CD11c+CD11b+), macrophages (CD45+MHCII+Ly6C–CD64+), and IL-17+ γδT cells (CD45+CD3+γδTCR+IL-17A+) (n = 5). (I) Statistical data of the skin infiltration of moDCs, macrophages, and IL-17+ γδT cells (n = 5). Scale bars: 100 μm (D). P values were determined by a 2-tailed unpaired t test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a; IMQ, imiquimod; moDC, monocyte-derived DC; PASI, Psoriasis Area and Severity Index.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Expressing, Flow Cytometry, Derivative Assay

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: (A) Schematic diagram for IMQ application and depletion of monocytes plus neutrophils or neutrophils alone in WT or miR-148a–/– mice. Phenotypic presentation (B) and H&E staining (D) of skin lesions of representative mice on day 5 (n = 3). Decrease in ear thickness (C, top), PASI score (C, bottom), dermal thickness (E, top), and acanthosis (E, bottom) on day 5 (n = 3). (F) The relative mRNA expression of indicated cytokines in skin (n = 3). (G) Statistical data of the skin infiltration of moDCs and IL-17+ γδ T cells (n = 3). Scale bars: 100 μm (D). P values were determined by a 2-tailed unpaired t test or by 1-way ANOVA with Bonferroni’s post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). moDC, monocyte-derived DC; miR-148a, microRNA-148a; PASI, Psoriasis Area and Severity Index.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Expressing, Derivative Assay

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: (A) Schematic diagram for IMQ application and intradermal administration of antagomir-NC or antagomir-148a. Phenotypic presentation (B) and H&E staining (D) of skin lesions of representative mice on day 5 (n = 6). Decrease in PASI score (C) and ear thickness (E) during the course of disease (n = 6). (F) The relative mRNA expression of indicated cytokines in skin (n = 6). Flow cytometry analysis (G) and statistical data (H) of the skin infiltration of moDCs and IL-17+ γδ T cells (n = 6). Scale bars: 100 μm (D). P values were determined by a 2-tailed unpaired t test or 1-way ANOVA with Bonferroni’s post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). IMQ, imiquimod; PASI, Psoriasis Area and Severity Index.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Expressing, Flow Cytometry

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: (A) Proinflammatory cytokine gene expression in the PBMCs derived from patients with psoriasis (n = 34) and healthy controls (n = 51). (B) Expression of miR-148a in peripheral monocytes isolated from patients with psoriasis (n = 34) and healthy controls (n = 51). (C) The correlation analysis between the miR-148a level in psoriatic monocytes and the mRNA level of IL23a and IL17a in psoriatic PBMCs (n = 34). (D and E) Expression of MAFB (D) and PU.1 (E) in peripheral monocytes isolated from patients with psoriasis (n = 20) and healthy controls (n = 23), and the correlation analysis between the miR-148a and MAFB (D) or PU.1 (E) level in psoriatic patients (n = 20). P values compare the indicated groups using nonparametric Mann-Whitney U test or Spearman’s test. Data are shown as mean ± SEM (*P < 0.05, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Gene Expression, Derivative Assay, Expressing, Isolation, MANN-WHITNEY

Journal: JCI Insight

Article Title: MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

doi: 10.1172/jci.insight.133721

Figure Lengend Snippet: miR-148a, which could be transcriptionally activated by PU.1, promotes moDC differ-entiation by directly targeting MAFB, thereby causing the immune imbalance and inflammatory response. miR-148a, microRNA-148a; moDC, monocyte-derived DC.

Article Snippet: The agomir-148a (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B06002","term_id":"1415280","term_text":"B06002"}} B06002 ) and the corresponding negative control (GenePharma, {"type":"entrez-nucleotide","attrs":{"text":"B04008","term_id":"1413286","term_text":"B04008"}} B04008 ) were purchased from Shanghai GenePharma Co. Ltd. Human PU.1 siRNA (Santa Cruz, sc-36330), mouse PU.1 siRNA (Santa Cruz, sc-36331), and MAFB siRNA (Santa Cruz, sc-35840) were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay