Huang Dan, Lou Cheng-Yu, Yu Ya-Lan, Fang Li
Graduate Collaborative Training Base of the First Hospital of Changsha, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China.
Department of Cardiology, The Affiliated Changsha Hospital of Xiangya School of Medicine, Central South University, Changsha, 410008, China.
Biochem Biophys Res Commun. 2025 Mar 25;754:151557. doi: 10.1016/j.bbrc.2025.151557. Epub 2025 Feb 27.
This study aims to investigate the role of nucleolin in the proliferation of vascular smooth muscle cells (VSMCs) and the underlying molecular mechanism, with a focus on protein-mRNA interactions. Various methods, including BrdU labeling, protein quantification, and CCK-8 cell proliferation assay, were employed to assess the effects of VSMCs proliferation induced by angiotensin II (Ang II) as well as the expressions of TGF-β1 and VEGF at the mRNA and protein levels. Gene transfection, RNA interference, and nucleolin mutant (Nucl-309) were utilized to examine the regulatory impact of nucleolin on the expression and the stability of the TGF-β1 and VEGF mRNA. Additionally, immunoprecipitation, RNA-EMSA, and luciferase reporter gene assays were conducted to investigate the binding of nucleolin to the 3' UTR of the TGF-β1 and VEGF mRNA. It was found that Ang II increased the DNA synthesis capacity, total cell protein content, and cell survival rate of VSMCs, and the expressions of TGF-β1 and VEGF gradually increased upon stimulation by Ang II. Nucleolin overexpression and knockdown significantly enhanced and inhibited the expressions of TGF-β1 and VEGF, respectively. The overexpression of the nucleolin mutant showed no regulatory effect on the expressions of TGF-β1 and VEGF. The interaction between nucleolin and the 3' UTR of the TGF-β1 and VEGF mRNA increased their stability and boosted their expressions. Hence, nucleolin plays a key role in promoting Ang II-induced VSMCs proliferation by enhancing the stability of the TGF-β1 and VEGF mRNA through binding to their respective 3' UTR, which ultimately upregulates their protein expression.
本研究旨在探讨核仁素在血管平滑肌细胞（VSMC）增殖中的作用及其潜在分子机制，重点关注蛋白质与mRNA的相互作用。采用了多种方法，包括BrdU标记、蛋白质定量和CCK-8细胞增殖测定，以评估血管紧张素II（Ang II）诱导的VSMC增殖效应以及TGF-β1和VEGF在mRNA和蛋白质水平的表达。利用基因转染、RNA干扰和核仁素突变体（Nucl-309）来检测核仁素对TGF-β1和VEGF mRNA表达及稳定性的调节作用。此外，进行了免疫沉淀、RNA-EMSA和荧光素酶报告基因测定，以研究核仁素与TGF-β1和VEGF mRNA的3'UTR的结合情况。结果发现，Ang II增加了VSMC的DNA合成能力、总细胞蛋白含量和细胞存活率，并且在Ang II刺激后TGF-β1和VEGF的表达逐渐增加。核仁素的过表达和敲低分别显著增强和抑制了TGF-β1和VEGF的表达。核仁素突变体的过表达对TGF-β1和VEGF的表达没有调节作用。核仁素与TGF-β1和VEGF mRNA的3'UTR之间的相互作用增加了它们的稳定性并促进了它们的表达。因此，核仁素通过与TGF-β1和VEGF各自的3'UTR结合来增强其mRNA的稳定性，从而在促进Ang II诱导的VSMC增殖中起关键作用，最终上调它们的蛋白质表达。
