Journal: Frontiers in Immunology

Article Title: Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a

doi: 10.3389/fimmu.2020.00491

Figure Lengend Snippet: Estrogen impairs HDI mediated upregulation of Aicda- targeting miR-26a expression. (A) Alignment of miR-181b, miR-26a, miR-125a, miR-155, miR-103, miR-361, and miR-92b with their target sites in the 3′ UTR of Aicda mRNA ( Aicda 3′UTR has two mR-181b target sites). (B) SCFA HDI butyrate upregulates Aicda- targeting miR-155, miR-181a, miR-361, miR-92b, miR-125a, and miR-26a, while estrogen downregulates miR-26a and reverses SCFA-mediated upregulation of this miRNA. B cells were stimulated with LPS plus IL-4 and treated with nil, E2 (30 nM), butyrate (500 μM) or butyrate (500 μM) plus E2 (30 nM) for 60 h. Expression of miRNAs was analyzed by qRT-PCR, normalized to expression of small nuclear/nucleolar RNAs Rnu6, Snord61, Snord68, and Snord70, and depicted as relative to the expression of this miRNA in B cells treated with nil, set as 1. Data are mean ± SEM from three independent experiments. (C) Expression of miR-125a and miR-26a in B cells stimulated with LPS plus IL-4 for 60 h, as analyzed by miRNA-Seq and depicted as RPK. Data are means ± SEM of three independent experiments. (D) miR-26a is upregulated by butyrate and downregulated by E2 in CH12F3 B cells. CH12F3 cells were stimulated with CD154 plus IL-4 and TGF-β, and treated with nil, E2 (30 nM), butyrate (500 μM) or butyrate (500 μM) plus E2 (30 nM) for 60 h. Expression of miRNA-26a was analyzed by qRT-PCR, normalized to expression of Snord70, and depicted as relative to the expression of this miRNA in CH12F3 cells treated with nil, set as 1. Data are mean ± SEM from three independent experiments. (E) SCFA and E2 modulate miRNAs that target Aicda 3′UTR. Luciferase activity in CH12F3 cells transfected with Aicda 3′UTRs (cloned into pMIR-REPORT luciferase reporter vector) after a 24-h treatment with nil, E2 (30 nM), butyrate (500 μM) or butyrate (500 μM) plus E2 (30 nM). Luciferase activity was measured 1.5 h after transfection. Transfection efficiency was controlled by normalizing to signal from co-transfected R. reniformis luciferase vector. Luciferase activity is depicted as relative to values in B cells cultured with nil, set as 100. Data are mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, ns, not significant (unpaired t -test).

Article Snippet: The miR-26a expression retroviral construct pMSCV-PIG-miR-26a-2 ( ) was a gift from Joshua Mendell (Addgene plasmid # 64230), pMSCV PIG (Puro IRES GFP empty vector) was a gift from David Bartel (Addgene plasmid # 21654).

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Clone Assay, Plasmid Preparation, Cell Culture

Journal: Frontiers in Immunology

Article Title: Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a

doi: 10.3389/fimmu.2020.00491

Figure Lengend Snippet: MIR-26a modulates Aicda expression and CSR. (A) Luciferase activity in CH12F3 cells transfected with wild-type or mutated Aicda 3′UTRs (cloned into pMIR-REPORT luciferase reporter vector) after a 24-h treatment with Nil, E2 (30 nM), butyrate (500 μM) or butyrate (500 μM) plus E2 (30 nM). Luciferase activity was measured 1.5 h after transfection. Transfection efficiency was controlled for by normalizing to signal from co-transfected R. reniformis luciferase vector. Luciferase activity is depicted as relative to values in B cells cultured with nil, set as 100. Data are mean ± SEM from three independent experiments. (B) Enforced expression of miR-26a reduced Aicda . B cells isolated from C57BL/6 mice were transduced with pMSCV-PIG-miR-26a-2 retroviral vector expressing GFP and miR-26a or empty pMSCV-PIG retroviral vector that expression GFP, and cultured for 48 h. Expression of Aicda was analyzed by real-time qRT-PCR, normalized to Gapdh , and depicted as relative to the expression of Aicda in B cells transduced with empty pMSCV-PIG retroviral vector, set as 1. Data are mean ± SEM from three independent experiments. (C) Enforced expression of miR-26a reduced CSR. Proportions of surface IgG1 + B cells among empty pMSCV-PIG or pMSCV-PIG-miR-26a-2 retroviral vector-transduced (B220 + GFP + ) B cells were analyzed by flow cytometry 96 h after transduction. Data are from one representative of three independent experiments. (D) Sequence of miR-26a sponge which contains five repeats of miRNA antisense binding sites (indicated by red boxes); sequences complimentary to mature miR-26a are underlined. (E,F) Expression of miR-26a sponge increased Aicda expression. B cells were transduced with pMG-miR-26a-sponge retroviral vector (expressing GFP and miR-26a sponge) or empty pMG retroviral vector (expressing GFP), and cultured for 48 h. Expression of Aicda was analyzed by real-time qRT-PCR, normalized to Gapdh , and depicted as relative to the expression of Aicda in B cells transduced with empty pMSCV-PIG retroviral vector, set as 1. Data are mean ± SEM from three independent experiments (E) . (F) AID and β-Actin proteins as analyzed by immune-blotting. (G) Expression of miR-26a sponge increased CSR. Proportions of surface IgG1 + B cells among empty pMG or pMG-miR-26a-sponge retroviral vector-transduced (B220 + GFP + ) B cells, as analyzed by flow cytometry 96 h after transduction. Data are from one representative of three independent experiments. * p < 0.05, ** p < 0.01, ns, not significant (unpaired t -test).

Article Snippet: The miR-26a expression retroviral construct pMSCV-PIG-miR-26a-2 ( ) was a gift from Joshua Mendell (Addgene plasmid # 64230), pMSCV PIG (Puro IRES GFP empty vector) was a gift from David Bartel (Addgene plasmid # 21654).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Clone Assay, Plasmid Preparation, Cell Culture, Isolation, Transduction, Retroviral, Quantitative RT-PCR, Flow Cytometry, Sequencing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a

doi: 10.3389/fimmu.2020.00491

Figure Lengend Snippet: B cells miR-26a is regulated by ERα upon ERα recruitment to the promoters of miR-26a host genes CTDSPL and CTDSP1 after E2 treatment. (A) Recruitment of ERα to the CTDSPL and CTDSP1 promoters in B cells upon E2 treatment. B cells were stimulated with LPS plus IL-4 in the absence or presence of E2 (0, 30, and 100 nM) for 48 h, as analyzed by ChIP assay and qPCR and normalized to the input. Data are mean ± SEM from three independent experiments. (B) Estrogen downregulates miR-26a expression. Esr1 +/+ and Esr1 −/− B cells were stimulated with LPS plus IL-4 and treated with nil, E2 (30 nM), butyrate (500 μM) or butyrate (500 μM) plus E2 (30 nM) for 60 h. Expression of miR-26a was analyzed by qRT-PCR, normalized to expression of small nuclear/nucleolar RNAs Rnu6, Snord61, Snord68, and Snord70, and depicted as relative to the expression of this miRNA in Esr1 +/+ B cells treated with nil, set as 1. Data are mean ± SEM from three independent experiments. (C) CH12F3 cells were stimulated with CD154 plus IL-4 and TGF-β and treated with nil, E2 (30 nM), butyrate (500 μM) or butyrate (500 μM) plus E2 (30 nM) for 60 h. Expression of c-Myc transcripts was analyzed by qRT-PCR, normalized to expression of Gapdh , and depicted as relative to the expression of c-Myc in CH12F3 cells treated with nil, set as 1. Data are mean ± SEM from three independent experiments. (D) B cells were stimulated with LPS plus IL-4 in the absence or presence of E2 (0, 30, and 100 nM) for 60 h. Expression of c-Myc transcripts was analyzed by qRT-PCR, normalized to expression of Gapdh , and depicted as relative to the average expression level of c-Myc in B cells treated with nil, set as 1. Data are mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, ns, not significant (unpaired t test).

Article Snippet: The miR-26a expression retroviral construct pMSCV-PIG-miR-26a-2 ( ) was a gift from Joshua Mendell (Addgene plasmid # 64230), pMSCV PIG (Puro IRES GFP empty vector) was a gift from David Bartel (Addgene plasmid # 21654).

Techniques: Expressing, Quantitative RT-PCR