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MicroRNA-30b is a multifunctional regulator of aortic valve interstitial cells  Mi Zhang, MD, Xiaohong Liu, MD, Xiwu Zhang, MD, Zhigang Song, MD, Lin Han,

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Presentation on theme: "MicroRNA-30b is a multifunctional regulator of aortic valve interstitial cells  Mi Zhang, MD, Xiaohong Liu, MD, Xiwu Zhang, MD, Zhigang Song, MD, Lin Han,"— Presentation transcript:

1 MicroRNA-30b is a multifunctional regulator of aortic valve interstitial cells 
Mi Zhang, MD, Xiaohong Liu, MD, Xiwu Zhang, MD, Zhigang Song, MD, Lin Han, MD, Yuanyuan He, MD, Zhiyun Xu, MD  The Journal of Thoracic and Cardiovascular Surgery  Volume 147, Issue 3, Pages e2 (March 2014) DOI: /j.jtcvs Copyright © Terms and Conditions

2 Figure 1 MicroRNA-30b (miRNA-30b) is downregulated in osteogenic valve interstitial cells. A, Relative expression levels of microRNA-30 types (miRNA-30s) in calcified regions were determined by quantitative real-time polymerase chain reaction (n = 10, mean ± SD). Asterisk indicates P < .05. B, MicroRNA-30b levels were significantly reduced in bone morphogenetic protein 2 (BMP-2)–treated cells in 5 independent replications (P < .05). The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

3 Figure 2 MicroRNA-30b inhibits bone morphogenetic protein 2 (BMP-2) signaling–induced osteogenesis in cultured valve interstitial cells. A, Alkaline phosphatase (ALP) activity of 5 independent experiments in each group (mean ± SD). Compared with other groups, microRNA-30b inhibitor (I-miRNA-30b) activated alkaline phosphatase to a greater extent. Asterisk indicates P < .05. B, The levels of osteocalcin messenger RNA (mRNA) were determined by quantitative real-time polymerase chain reaction (mean ± SD). Inhibition of microRNA-30b upregulated osteocalcin messenger RNA. Asterisk indicates P < .05. C, On immunoblotting, miRNA-30b was seen to have an obvious effect on osteocalcin protein expression. miRNA-NC, microRNA negative control; M-miRNA-30b, microRNA-30b mimic. The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

4 Figure 3 MicroRNA-30b suppresses apoptosis in valve interstitial cells. Apoptotic cells are present in the upper right (early stage), and lower right (late stage) quadrants of each panel. The most significant increase in the proportion of apoptotic valve interstitial cells was observed in cells transfected with microRNA-30b inhibitor (I-miRNA-30b). In contrast, valve interstitial cells treated with microRNA-30b mimic (M-miRNA-30b) showed decreased apoptosis. BMP-2, Bone morphogenetic protein 2; miRNA-NC, microRNA negative control. The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

5 Figure 4 MicroRNA-30b attenuates bone morphogenetic protein 2 (BMP-2)–induced osteogenic differentiation in valve interstitial cells. Valve interstitial cells were evaluated with regard to micro RNA-30b. After culture in osteogenic differentiation medium for 4 days, quantitative real-time polymerase chain reaction and Western blotting were performed to evaluate osteogenic differentiation. A, MicroRNA-30b regulated the osteoblast markers Runx2, Smad1, and caspase-3 at the messenger RNA (mRNA) level. Asterisk indicates P < .05. B, Western blots for Runx2, Smad1, p-Smad1, and caspase-3 were performed with the total cell lysates. miRNA-NC, microRNA negative control; M-miRNA-30b, microRNA-30b mimic; I-miRNA-30b, microRNA-30b inhibitor. The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

6 Figure 5 Runx2, Smad1, and caspase-3 are direct targets of microRNA-30b (miRNA-30b). A, Schematic representation of putative target sites for microRNA-30b in the 3′ untranslated regions (UTRs) of human Runx2, Smad1, and caspase-3 messenger RNA. B, Transfection with a micro RNA-30b mimic (M-miRNA-30b) inhibited Renilla-luciferase activity, whereas microRNA-30b knockdown with a microRNA-30b inhibitor (I-miRNA-30b) enhanced it. The Renilla-luciferase signal was normalized against the firefly-luciferase signal. The results are presented as the mean ± SD for each group of cells from 4 independent experiments. Dagger and asterisk indicate P < .05. miRNA-NC, MicroRNA negative control. The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

7 Figure E1 Identification of the primary aortic valve interstitial cells. A, Microscopic analysis of cell morphology. B, The primary aortic valve interstitial cells express specific markers (α-SMA 40%, vimentin 96%). The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

8 Figure E2 Histopathologic staining of the calcified aortic valves. Arrows indicate the positive immunohistochemical staining regions for Runx2 (A), osteocalcin (B), caspase-3 (C), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (D) in the high-magnification view (original magnification ×200). Bar represents 50 μm. The Journal of Thoracic and Cardiovascular Surgery  , e2DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

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