Chen Rong, Kong Peng, Zhang Fan, Shu Ya-Nan, Nie Xi, Dong Li-Hua, Lin Yan-Ling, Xie Xiao-Li, Zhao Li-Li, Zhang Xiang-Jian, Han Mei
Department of Biochemistry and Molecular Biology, College of Basic Medicine, Key Laboratory of Medical Biotechnology of Hebei Province, Key Laboratory of Neural and Vascular Biology of Ministry of Education, Hebei Medical University, Shijiazhuang 050017, PR China; Department of Neurology, Hebei Key Laboratory of Vascular Homeostasis and Hebei Collaborative Innovation Center for Cardio-cerebrovascular Disease, Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, PR China.
Department of Biochemistry and Molecular Biology, College of Basic Medicine, Key Laboratory of Medical Biotechnology of Hebei Province, Key Laboratory of Neural and Vascular Biology of Ministry of Education, Hebei Medical University, Shijiazhuang 050017, PR China.
Gene. 2017 Jun 15;616:52-57. doi: 10.1016/j.gene.2017.03.028. Epub 2017 Mar 23.
Recent studies have revealed that long non-coding RNAs (lncRNAs) participate in vascular homeostasis and pathophysiological conditions development. But still very few literatures elucidate the regulatory mechanism of non-coding RNAs in this biological process. Here we identified lncRNA taurine up-regulated gene 1 (TUG1) in rat vascular smooth muscle cells (VSMCs), and got 4612bp nucleotide sequence. The expression level of TUG1 RNA was increased in synthetic VSMCs by real-time PCR analysis. Meanwhile, the expression of enhancer of zeste homolog 2 (EZH2) (TUG1 binding protein) increased in cytoplasm of VSMCs under the same conditions. Immunofluoresce analysis displayed the colocalization of EZH2 with α-actin in cytoplasm and F-actin in cell edge ruffles. This leads us to hypothesize the existence of cytoplasmic TUG1/EZH2/α-actin complex. Using RNA pull down assay, we found that TUG1 interacted with both EZH2 and α-actin. Disruption of TUG1 abolished the interaction of EZH2 with α-actin, and accelerated depolymerization of F-actin in VSMCs. Based on EZH2 methyltransferase activity and the potential methylation sites in α-actin structure, we revealed that α-actin was lysine-methylated. Furthermore, the methylation of α-actin was inhibited by knockdown of TUG1. In conclusion, these findings partly suggested that EZH2-mediated methylation of α-actin may be dependent on TUG1, and thereby promotes cortex F-actin polymerization in synthetic VSMCs.
最近的研究表明，长链非编码RNA（lncRNAs）参与血管稳态和病理生理状况的发展。但仍很少有文献阐明非编码RNA在这一生物学过程中的调控机制。在此，我们在大鼠血管平滑肌细胞（VSMCs）中鉴定出lncRNA牛磺酸上调基因1（TUG1），并获得了4612bp的核苷酸序列。通过实时PCR分析发现，合成型VSMCs中TUG1 RNA的表达水平升高。同时，在相同条件下，VSMCs细胞质中zeste同源物2增强子（EZH2）（TUG1结合蛋白）的表达增加。免疫荧光分析显示EZH2与细胞质中的α-肌动蛋白以及细胞边缘褶皱中的F-肌动蛋白共定位。这使我们推测细胞质中存在TUG1/EZH2/α-肌动蛋白复合物。通过RNA下拉实验，我们发现TUG1与EZH2和α-肌动蛋白均相互作用。破坏TUG1可消除EZH2与α-肌动蛋白的相互作用，并加速VSMCs中F-肌动蛋白的解聚。基于EZH2甲基转移酶活性以及α-肌动蛋白结构中的潜在甲基化位点，我们发现α-肌动蛋白发生了赖氨酸甲基化。此外，敲低TUG1可抑制α-肌动蛋白的甲基化。总之，这些发现部分表明EZH2介导的α-肌动蛋白甲基化可能依赖于TUG1，从而促进合成型VSMCs中皮质F-肌动蛋白的聚合。
