MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts

• Published in: Nan fang yi ke da xue xue bao = Journal of Southern Medical University 2018-09, Vol.38 (10), p.1203-1208
• Main Authors: Liang, Jingnan, Zhu, Wensi, Zhang, Zhuo, Zhu, Jiening, Fu, Yongheng, Lin, Qiuxiong, Kuang, Sujuan, Zhang, Mengzhen, Shan, Zhixin
• Format: Article
• Language: Chinese
• Subjects: 3' Untranslated Regions; Animals; Fibroblasts - metabolism; Fibrosis; Genes, Reporter; Luciferases - genetics; Mice; Mice, Inbred C57BL; MicroRNAs - genetics; MicroRNAs - metabolism; RNA Interference; RNA, Small Interfering - genetics; Smad1 Protein - genetics; Smad1 Protein - metabolism; Smad3 Protein - genetics; Smad3 Protein - metabolism; Transfection - methods
• ISSN: 1673-4254
• DOI: 10.3969/j.issn.1673-4254.2018.10.08

To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p. 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. 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. 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.