Jingnan Liang1, Wensi Zhu2, Zhuo Zhang3, Jiening Zhu3, Yongheng Fu3, Qiuxiong Lin3, Sujuan Kuang3, Mengzhen Zhang3, Zhixin Shan1,3. 1. School of Pharmacy, Southern Medical University, Guangzhou 510515, China. 2. Department of Pharmacy, Guangdong Women and Children's Hospital, Guangzhou 511400, China. 3. Research Center of Medical Sciences, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
Abstract
OBJECTIVE: To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p. METHODS: Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting. RESULTS: Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts. CONCLUSIONS: As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.
OBJECTIVE: To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p. METHODS: Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting. RESULTS: Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts. CONCLUSIONS: As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.
Authors: Xiang-Yang Zhu; Elena Daghini; Martin Rodriguez-Porcel; Alejandro R Chade; Claudio Napoli; Amir Lerman; Lilach O Lerman Journal: Atherosclerosis Date: 2006-09-22 Impact factor: 5.162
Authors: Wansheng Wang; Xiao R Huang; Ellery Canlas; Kazuhiro Oka; Luan D Truong; Chuxia Deng; Neil A Bhowmick; Wenjun Ju; Erwin P Bottinger; Hui Y Lan Journal: Circ Res Date: 2006-03-23 Impact factor: 17.367