miR-181b targets semaphorin 3A to mediate TGF-β–induced endothelial-mesenchymal transition related to atrial fibrillation
Ying-Ju Lai, Feng-Chun Tsai, Gwo-Jyh Chang, Shang-Hung Chang, Chung-Chi Huang, Wei-Jan Chen, Yung-Hsin Yeh
Ying-Ju Lai, Feng-Chun Tsai, Gwo-Jyh Chang, Shang-Hung Chang, Chung-Chi Huang, Wei-Jan Chen, Yung-Hsin Yeh
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Research Article Cardiology

miR-181b targets semaphorin 3A to mediate TGF-β–induced endothelial-mesenchymal transition related to atrial fibrillation

Abstract

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β–treated human AEECs, cardiac-specific TGF-β–transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β–treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β–transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.

Authors

Ying-Ju Lai, Feng-Chun Tsai, Gwo-Jyh Chang, Shang-Hung Chang, Chung-Chi Huang, Wei-Jan Chen, Yung-Hsin Yeh

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Figure 4

miR-181b targets Sema3A, and TGF-β induces Twist and SMA expression.

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miR-181b targets Sema3A, and TGF-β induces Twist and SMA expression. (A)...
(A) Representative immunoblots showing protein expression in AEECs treated with 5 ng/mL TGF-β for up to 48 hours. GAPDH was used as the loading control. (B) Representative immunoblots showing protein expression in AEECs incubated with or without TGF-β (0, 1, 2, or 5 ng/mL). (C) Representative immunoblots show protein expression in AEECs that were transfected with miR-181b antagomir (antagomir-181b) or scrambled control miRNA and then treated with or without TGF-β (5 ng/mL). (D) Confirmation of the hsa-miR-181b target site in the Sema3A 3′-UTR. Schematic representation of the Sema3A 3′-UTR indicating the predicted hsa-miR-181b binding site. AEECs were transfected with the pMIR-REPORT-Sema3A 3′-UTR (intact) or pMIR-REPORT-Sema3A 3′-UTR (mutant) luciferase reporter vector. The fold induction in relative luciferase activity was plotted (n = 3). *P < 0.05 compared with the scrambled control, by 2-tailed Student’s t test. (E) AEECs were transfected with the miR-181b TSB for 24 hours, after which AEECs were treated with or without TGF-β for 6 hours. Sema3A mRNA expression was measured by qRT-PCR (n = 3). *P < 0.05 compared with control; #P < 0.05 and ###P < 0.001 compared with TGF-β; 1-way ANOVA with Bonferroni’s post hoc test. (F) AEECs were transfected with the negative control mimic (NC mimic) or the synthetic miR-181b mimic, with or without the miR-181b TSB. Sema3A mRNA expression was measured by qRT-PCR (n = 3). **P < 0.01 compared with NC mimic; ##P < 0.01 compared with miR-181b mimic; 1-way ANOVA with Bonferroni’s post hoc test. (G) Representative quantitative data for the immunoblots showing protein expression in AEECs transfected with miR-181b TSBs for 24 hours (n = 3). ***P < 0.001 versus the NC mimic group; ###P < 0.001 versus the 181b mimic group; 1-way ANOVA with Bonferroni’s post hoc test. (H) AEECs were transfected with Sema3A siRNA or scrambled siRNA. Representative immunoblots and densitometric quantification of protein expression are shown (n = 3). **P < 0.01 and ***P < 0.001 compared with scrambled siRNA–treated group, by 2-tailed Student’s t test. t, total.