miR-27a-3p Targets ATF3 to Reduce Calcium Deposition in Vascular Smooth Muscle Cells

Abstract

Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor of morbidity and mortality associated with these diseases. Herein, we report that reduction of microRNA-27a-3p (miR-27a-3p) causes an increase in activating transcription factor 3 (ATF3), a novel osteogenic transcription factor, in vascular smooth muscle cells. Both microRNA (miRNA) and mRNA microarrays were performed with rat vascular smooth muscle cells, and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatics analysis. Inorganic phosphate significantly reduced the expression of miR-27a-3p in A10 cells. The transcript level was also reduced in vitamin D₃-administered mouse aortas. miR-27a-3p mimic reduced calcium deposition, whereas miR-27a-3p inhibitor increased it. The Atf3 mRNA level was upregulated in a cellular vascular calcification model, and miR-27a-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-27a-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-27a-3p may contribute to the development of vascular calcification by de-repression of ATF3.

Keywords: activating transcription factor 3; miR-27a-3p; miRNA; vascular calcification; vascular smooth muscle cells.

Graphical abstract

Figure 1

Strategies for the Discovery of Novel miRNA/Target Gene Sets and Confirmation of Changes of miR-27a-3p (A) Strategy to find a novel miRNA and its target. Primarily cultured rat vascular smooth muscle cells (rVSMCs) were treated for 6 days and total RNA was prepared for a miRNA microarray and mRNA microarray. The samples were duplicated for each condition. The averaged value was used for analysis. (B) Gene tree analysis with the miRNA microarray results (GEO: GSE130486). (C) Changes in miR-27a-3p in microarray results in rVSMCs. Two values were averaged. (D) qRT-PCR analysis. Inorganic phosphate (Pi) treatment for 6 days significantly reduced the miR-27a-3p transcript amount in A10 cells. (E) Time course of Pi treatment and the decrease in miR-27a-3p amount. (F) The miR-27a-3p transcript level was significantly downregulated in the aorta obtained from vitamin D₃-administered mice. Error bars indicate SD. ∗p < 0.05, ∗∗p < 0.01.

Figure 2

Pi-Induced Calcium Deposition Was Significantly Attenuated by Transfection with miR-27-a-3p mimic (A) Pi (6 days) significantly increased the calcium deposition in A10 cells, a rVSMC line. However, the increase was significantly blunted when miR-27a-3p mimic was transfected. (B) Pi (2 days)-induced increase in calcium deposition was potentiated by simultaneous transfection of miR-27a-3p inhibitor. (C) Alizarin red staining to show A10 mineralization. miR-27a-3p mimic blocked the Pi-induced increase in the calcium deposition. (D) The increase in the transcript level of Runx2, a key transcription factor of ossification, was completely abolished when miR-27a-3p mimic was co-transfected. (E) The increase in alkaline phosphatase (Alp) transcript level was also blocked by miR-27a-3p mimic transfection. (F) Western blot analysis to examine the Runx2 and Alp protein amount after transfection of miR-27a-3p mimic. (G and H) Quantification results of western blot analysis of Runx2 (G) and ALP (H) in the presence of Pi. Error bars indicate SD. ∗p < 0.05, ^@p < 0.05, ∗∗p < 0.01. NS, not significant.

Figure 3

miR-27a-3p Targets the 3′ UTR of ATF3 (A) Atf3 transcript level in primarily cultured rVSMCs treated with Pi. The results were obtained from an mRNA microarray (GEO: GSE74755). (B) miR-27a-3p mimic significantly attenuated the luciferase activity driven by Atf3-3′ UTR. psiCHECK2-Atf3-3′ UTR was used for luciferase activity measurement. (C) miR-27a-3p inhibitor dramatically increased the luciferase activity. (D) miR-27a-3p mimic failed to inhibit mutant Atf3-3′ UTR where the miR-27a-3p-binding sequence was altered. (E) miR-27a-3p mimic significantly reduced the mRNA transcript amount of Atf3. (F and G) miR-27a-3p mimic reduced the protein amount of Atf3 as determined by western blot analysis. Quantification results from the western blot (F) are shown in (G). Error bars indicate SD. ∗p < 0.05, ∗∗p < 0.01. NS, not significant.

Figure 4

Pi Induces Atf3 Expression, Which Is Attenuated by miR-27a-3p (A and B) Pi (6 days) increased the Atf3 transcript amount as determined by quantitative RT-PCR (A) or by western blot analysis (B). (C) The Atf3 transcript amount was gradually increased by Pi in a time-dependent fashion. (D) Atf mRNA was also increased by vitamin D₃ administration in mice. Numbers in bars represent the numbers of mice tested. Error bars indicate SD. ∗p < 0.05, ∗∗p < 0.01.

Figure 5

Histologic Analysis of miR-27a-3p and Atf3 in Vitamin D₃-Administered Mouse Aorta (A) Alizarin red S staining shows the marked increase in calcification. (B) In situ hybridization analysis shows the miR-27a-3p transcript. Digoxin signals shown in dark blue are high in the muscle tissue as well as in the pericyte of the vehicle-administered (0 d) mice. However, the expression in the smooth muscle tissue was gradually downregulated by administration of vitamin D₃. (C) Fluorescent immunohistochemistry analysis shows the expression of smooth muscle α-actin (green) and Atf3 (red). Note that the expression of α-smooth muscle actin was gradually decreased, whereas that of Atf3 was increased by day 6 of vitamin D₃ administration.

Figure 6

Atf3 Induces Calcium Deposition (A and B) Transfection of Atf3 increased calcium deposition. (A) Alizarin red S staining. (B) Calcium deposition. (C and D) Atf3 siRNA significantly reduced the calcium deposition in the presence of Pi. (C) Alizarin red S staining. (D) Atf3 siRNA significantly reduced the calcium contents in the presence of Pi. (E) Transfection of Atf3 induced an increase in the Runx2 mRNA level. (F) Promoter analysis. Atf3 increased the Runx2 P2 (proximal) promoter (right panel) in a dose-dependent fashion. Error bars indicate SD. ∗p < 0.05 ∗∗p < 0.01.

Figure 7

Anti-Calcification Effect of miR-27a-3p Is Blocked by Atf3 (A) Effect of miR-27a-3p mimic on Atf3 transcript amount. The Pi-induced increase as well as the basal level of Atf3 mRNA were significantly attenuated by transfection of miR-27a-3p mimic. (B) miR-27a-3p mimic-induced reduction of calcium deposition in the presence of Pi was completely blocked when Atf3 was overexpressed. (C and D) Changes in mRNA levels of Runx2 (C) and Alp (D). (E and F) miR-27a-3p mimic-induced reduction of Runx2 protein amount was recovered by co-transfection of pcDNA6-Atf3-myc. Quantitative results of (E) was shown in (F). (G and H) miR-27a-3p inhibitor-induced increase in Runx2 protein amount was blocked by simultaneous transfection of Atf3 siRNA. Quantitative results of (G) are shown in (H). (I) Diagram. Pi reduces the expression of miR-27a-3p, which results in the activation of ATF3, a novel pro-calcific mediator. Error bars indicate SD. ∗p < 0.05, ∗∗p < 0.01, ^@@p < 0.01. NS, not significant.