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  • Title: [MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts].
    Author: Liang J, Zhu W, Zhang Z, Zhu J, Fu Y, Lin Q, Kuang S, Zhang M, Shan Z.
    Journal: Nan Fang Yi Ke Da Xue Xue Bao; 2018 Sep 30; 38(10):1203-1208. PubMed ID: 30377137.
    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. 目的: 探讨微小RNA-199a-3p（miR-199a-3p）对心肌纤维化的调控作用及其作用靶基因。 方法: 原代分离并体外培养成体C57BL/6小鼠心肌成纤维细胞；脂质体转染法将miR-199a-3p模拟物和Smad1 siRNA瞬时转染至小鼠心肌成纤维细胞；双荧光素酶报告基因实验检测miR-199a-3p与潜在靶基因Smad1 3'端非翻译区（3'-UTR）的结合作用；实时荧光定量PCR（RT-qPCR）和Western blot法分别检测小鼠心肌成纤维细胞转染miR-199a-3p后，Smad1及纤维化相关基因表达。Western blot检测转染miR-199a-3p，Smad1 siRNA后，小鼠心肌成纤维细胞中纤维化相关基因、Smad1和Smad3的激活水平。 结果: RT-qPCR和Western blot结果证实在心肌成纤维细胞中过表达miR-199a-3p可以增强纤维化相关基因表达。双荧光素酶报告基因实验显示miR-199a-3p与Smad1 3'-UTR有结合作用。RT-qPCR和Western blot结果证实miR-199a-3p可在转录水平抑制Smad1表达。过表达miR-199a-3p和沉默Smad1均能一致性的通过激活Smad3通路而促进心肌成纤维细胞中纤维化相关基因表达。 结论: miR-199a-3p通过靶向Smad1，从而激活Smad3通路来促进纤维化相关基因表达。
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