[1]梁景南,朱文思,张灼,等.miR-199a-3p靶向Smad1促进小鼠心肌纤维化相关基因的表达[J].南方医科大学学报,2018,(10):1203.[doi:10.12122/j.issn.1673-4254.2018.10.08]
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miR-199a-3p靶向Smad1促进小鼠心肌纤维化相关基因的表达()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2018年10期
页码:
1203
栏目:
出版日期:
2018-10-16

文章信息/Info

Title:
MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts
作者:
梁景南朱文思张灼朱杰宁符永恒林秋雄邝素娟张梦珍单志新
关键词:
心肌纤维化心肌成纤维细胞微小RNASmad1
Keywords:
cardiac fibrosis cardiac fibroblasts microRNA Smad1
DOI:
10.12122/j.issn.1673-4254.2018.10.08
摘要:
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。
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.

相似文献/References:

[1]马新英,杨明会,陈劲松,等.补肾活血方对大鼠心脏成纤维细胞增殖及胶原表达的影响[J].南方医科大学学报,2012,(01):122.
[2]李芳,曾欧,罗健,等.硫化氢对糖尿病大鼠心肌纤维化及MAPK1/3和MMP-8表达的影响[J].南方医科大学学报,2015,(04):549.
[3]王世祥,陆志锋,许卫,等.依达拉奉可减轻大鼠氧化应激及延缓心肌纤维化[J].南方医科大学学报,2015,(11):1591.

更新日期/Last Update: 1900-01-01