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The role of microRNA-1246 in the regulation of B cell activation and the pathogenesis of systemic lupus erythematosus.

Luo S, Liu Y, Liang G, Zhao M, Wu H, Liang Y, Qiu X, Tan Y, Dai Y, Yung S, Chan TM, Lu Q - Clin Epigenetics (2015)

Bottom Line: Our results showed that the expression of miR-1246 was significantly decreased in B cells from SLE patients.We also observed that abnormal activation of the AKT signaling pathway was associated with decreased P53 expression, leading to the downregulation of the miR-1246 expression; and upregulation of the miR-1246 expression reversed the responsiveness of B cells by inhibiting EBF1 expression.Activated B cells in lupus could decrease the expression of miR-1246 through the AKT-P53 signaling pathway, which in turn enhances the expression of EBF1, thereby promoting further activation of B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Medical Epigenomics, Changsha, Hunan 410011 China.

ABSTRACT

Background: The pathogenesis of systemic lupus erythematosus (SLE) has not yet been completely elucidated. One of the hallmarks of SLE is the production of autoantibodies by uncontrolled over-activated B cells. Early B cell factor 1 (EBF1) contributes to the development, activation, and proliferation of B cells through activation of the AKT signaling pathway. Accumulating evidence has demonstrated that several microRNAs (miRNAs) contribute to the pathogenesis of autoimmune diseases through the regulation of B cells in SLE. We aim to investigate the expression patterns of miR-1246 in B cells and its contribution to pathogenesis of SLE.

Results: Our results showed that the expression of miR-1246 was significantly decreased in B cells from SLE patients. We verified that miR-1246 specifically targeted the EBF1 messenger RNA (mRNA) by interacting with its 3'-untranslated region (3'-UTR) and regulated the expression of EBF1. Transfection of miR-1246 inhibitors into healthy B cells upregulated the expression of EBF1, enhanced B cell function, and increased the production of B cell surface co-stimulatory molecules CD40, CD80, and CD86. We also observed that abnormal activation of the AKT signaling pathway was associated with decreased P53 expression, leading to the downregulation of the miR-1246 expression; and upregulation of the miR-1246 expression reversed the responsiveness of B cells by inhibiting EBF1 expression.

Conclusions: Activated B cells in lupus could decrease the expression of miR-1246 through the AKT-P53 signaling pathway, which in turn enhances the expression of EBF1, thereby promoting further activation of B cells. Conversely, upregulation of miR-1246 could interrupt this amplification pathway. Our findings thus provide a theoretical framework towards the research of novel biological targets in SLE treatment.

No MeSH data available.


Related in: MedlinePlus

Verification of miR-1246 target genes. (A-C) The expression levels of miR-1246, miR-126, miR-142-3p, and miR-142-5p (A) and early B cell factor 1 (EBF1) protein (B, C) were analyzed after transfection with miR-1246 inhibitor or inhibitor control. (D-F) The expression levels of miR-1246, miR-126, miR-142-3p, miR-142-5p (D), and EBF1 protein (E, F) were analyzed after transfection with the miR-1246 mimic or mimic control. Bars show the mean ± SD results in three healthy donors or three patients with active SLE. All experiments were performed in triplicate. The Western blot image is a representative image (n = 3). (G) Schematic representation of the EBF1 luciferase reporter construct is shown. The sequence of the miR-1246 binding site in the 3′-untranslated region (3′-UTR) of EBF1 (gray box) is shown on the left. Mutated residues are shown in red. (H) Relative firefly luciferase activity in Jurkat cells co-transfected with an empty vector (mimic control) or an miR-1246 mimic, together with luciferase reporter constructs containing either a wild-type (WT) or a mutated (Mut) EBF1 3′-UTR are shown. Values in (H) are the mean ± SD results from three independent experiments. (**P < 0.01).
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Fig3: Verification of miR-1246 target genes. (A-C) The expression levels of miR-1246, miR-126, miR-142-3p, and miR-142-5p (A) and early B cell factor 1 (EBF1) protein (B, C) were analyzed after transfection with miR-1246 inhibitor or inhibitor control. (D-F) The expression levels of miR-1246, miR-126, miR-142-3p, miR-142-5p (D), and EBF1 protein (E, F) were analyzed after transfection with the miR-1246 mimic or mimic control. Bars show the mean ± SD results in three healthy donors or three patients with active SLE. All experiments were performed in triplicate. The Western blot image is a representative image (n = 3). (G) Schematic representation of the EBF1 luciferase reporter construct is shown. The sequence of the miR-1246 binding site in the 3′-untranslated region (3′-UTR) of EBF1 (gray box) is shown on the left. Mutated residues are shown in red. (H) Relative firefly luciferase activity in Jurkat cells co-transfected with an empty vector (mimic control) or an miR-1246 mimic, together with luciferase reporter constructs containing either a wild-type (WT) or a mutated (Mut) EBF1 3′-UTR are shown. Values in (H) are the mean ± SD results from three independent experiments. (**P < 0.01).

Mentions: According to the TargetScan and miRBase bioinformatic software, EBF1, which is required for the proliferation, survival, and signaling of pro-B cells and peripheral B cell subsets, including B1 cells and marginal zone B cells [30], is a predicted target of miR-1246. To better understand the relationship between miR-1246 and EBF1, we plotted miR-1246 expression levels (measured by real-time RT-PCR) from individual SLE B cell lysates (n = 30) against EBF1 protein levels (measured by Western blotting) from the same samples (Figure 2A, B). In this case, a strong inverse correlation was observed (Figure 2C). To explore whether miR-1246 directly regulates EBF1, we transfected primary B cells from three healthy donors with a miR-1246 inhibitor or a control miRNA. Two days after transfection, a 3.45-fold reduction of miR-1246 level was observed while levels of the unrelated miR-126, miR-142-3p, and miR-142-5p remained unchanged (Figure 3A), and the level of EBF1 protein was significantly increased compared with negative controls (Figure 3B, C). Consistent with this finding, transfection of a miR-1246 mimic into SLE B cells induced a 3.72-fold upregulation of miR-1246 expression while those of the unrelated miR-126, miR-142-3p, and miR-142-5p remained unchanged (Figure 3D) and a significantly decreased level of EBF1 protein (Figure 3E, F).Figure 2


The role of microRNA-1246 in the regulation of B cell activation and the pathogenesis of systemic lupus erythematosus.

Luo S, Liu Y, Liang G, Zhao M, Wu H, Liang Y, Qiu X, Tan Y, Dai Y, Yung S, Chan TM, Lu Q - Clin Epigenetics (2015)

Verification of miR-1246 target genes. (A-C) The expression levels of miR-1246, miR-126, miR-142-3p, and miR-142-5p (A) and early B cell factor 1 (EBF1) protein (B, C) were analyzed after transfection with miR-1246 inhibitor or inhibitor control. (D-F) The expression levels of miR-1246, miR-126, miR-142-3p, miR-142-5p (D), and EBF1 protein (E, F) were analyzed after transfection with the miR-1246 mimic or mimic control. Bars show the mean ± SD results in three healthy donors or three patients with active SLE. All experiments were performed in triplicate. The Western blot image is a representative image (n = 3). (G) Schematic representation of the EBF1 luciferase reporter construct is shown. The sequence of the miR-1246 binding site in the 3′-untranslated region (3′-UTR) of EBF1 (gray box) is shown on the left. Mutated residues are shown in red. (H) Relative firefly luciferase activity in Jurkat cells co-transfected with an empty vector (mimic control) or an miR-1246 mimic, together with luciferase reporter constructs containing either a wild-type (WT) or a mutated (Mut) EBF1 3′-UTR are shown. Values in (H) are the mean ± SD results from three independent experiments. (**P < 0.01).
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Related In: Results  -  Collection

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Fig3: Verification of miR-1246 target genes. (A-C) The expression levels of miR-1246, miR-126, miR-142-3p, and miR-142-5p (A) and early B cell factor 1 (EBF1) protein (B, C) were analyzed after transfection with miR-1246 inhibitor or inhibitor control. (D-F) The expression levels of miR-1246, miR-126, miR-142-3p, miR-142-5p (D), and EBF1 protein (E, F) were analyzed after transfection with the miR-1246 mimic or mimic control. Bars show the mean ± SD results in three healthy donors or three patients with active SLE. All experiments were performed in triplicate. The Western blot image is a representative image (n = 3). (G) Schematic representation of the EBF1 luciferase reporter construct is shown. The sequence of the miR-1246 binding site in the 3′-untranslated region (3′-UTR) of EBF1 (gray box) is shown on the left. Mutated residues are shown in red. (H) Relative firefly luciferase activity in Jurkat cells co-transfected with an empty vector (mimic control) or an miR-1246 mimic, together with luciferase reporter constructs containing either a wild-type (WT) or a mutated (Mut) EBF1 3′-UTR are shown. Values in (H) are the mean ± SD results from three independent experiments. (**P < 0.01).
Mentions: According to the TargetScan and miRBase bioinformatic software, EBF1, which is required for the proliferation, survival, and signaling of pro-B cells and peripheral B cell subsets, including B1 cells and marginal zone B cells [30], is a predicted target of miR-1246. To better understand the relationship between miR-1246 and EBF1, we plotted miR-1246 expression levels (measured by real-time RT-PCR) from individual SLE B cell lysates (n = 30) against EBF1 protein levels (measured by Western blotting) from the same samples (Figure 2A, B). In this case, a strong inverse correlation was observed (Figure 2C). To explore whether miR-1246 directly regulates EBF1, we transfected primary B cells from three healthy donors with a miR-1246 inhibitor or a control miRNA. Two days after transfection, a 3.45-fold reduction of miR-1246 level was observed while levels of the unrelated miR-126, miR-142-3p, and miR-142-5p remained unchanged (Figure 3A), and the level of EBF1 protein was significantly increased compared with negative controls (Figure 3B, C). Consistent with this finding, transfection of a miR-1246 mimic into SLE B cells induced a 3.72-fold upregulation of miR-1246 expression while those of the unrelated miR-126, miR-142-3p, and miR-142-5p remained unchanged (Figure 3D) and a significantly decreased level of EBF1 protein (Figure 3E, F).Figure 2

Bottom Line: Our results showed that the expression of miR-1246 was significantly decreased in B cells from SLE patients.We also observed that abnormal activation of the AKT signaling pathway was associated with decreased P53 expression, leading to the downregulation of the miR-1246 expression; and upregulation of the miR-1246 expression reversed the responsiveness of B cells by inhibiting EBF1 expression.Activated B cells in lupus could decrease the expression of miR-1246 through the AKT-P53 signaling pathway, which in turn enhances the expression of EBF1, thereby promoting further activation of B cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Medical Epigenomics, Changsha, Hunan 410011 China.

ABSTRACT

Background: The pathogenesis of systemic lupus erythematosus (SLE) has not yet been completely elucidated. One of the hallmarks of SLE is the production of autoantibodies by uncontrolled over-activated B cells. Early B cell factor 1 (EBF1) contributes to the development, activation, and proliferation of B cells through activation of the AKT signaling pathway. Accumulating evidence has demonstrated that several microRNAs (miRNAs) contribute to the pathogenesis of autoimmune diseases through the regulation of B cells in SLE. We aim to investigate the expression patterns of miR-1246 in B cells and its contribution to pathogenesis of SLE.

Results: Our results showed that the expression of miR-1246 was significantly decreased in B cells from SLE patients. We verified that miR-1246 specifically targeted the EBF1 messenger RNA (mRNA) by interacting with its 3'-untranslated region (3'-UTR) and regulated the expression of EBF1. Transfection of miR-1246 inhibitors into healthy B cells upregulated the expression of EBF1, enhanced B cell function, and increased the production of B cell surface co-stimulatory molecules CD40, CD80, and CD86. We also observed that abnormal activation of the AKT signaling pathway was associated with decreased P53 expression, leading to the downregulation of the miR-1246 expression; and upregulation of the miR-1246 expression reversed the responsiveness of B cells by inhibiting EBF1 expression.

Conclusions: Activated B cells in lupus could decrease the expression of miR-1246 through the AKT-P53 signaling pathway, which in turn enhances the expression of EBF1, thereby promoting further activation of B cells. Conversely, upregulation of miR-1246 could interrupt this amplification pathway. Our findings thus provide a theoretical framework towards the research of novel biological targets in SLE treatment.

No MeSH data available.


Related in: MedlinePlus