Center of Biochemistry Research, University of South China, Hengyang, Hunan, PR China.
PLoS One. 2013 Aug 5;8(8):e70658. doi: 10.1371/journal.pone.0070658. Print 2013.
This study was to investigate the molecular mechanisms underlying the 27nt-miRNA-mediated regulation of expression of the endothelial nitric oxide synthase (eNOS) gene.
Cell lines overexpressing 27nt-miRNA or its mutant were established by transfecting the miRNA expression vector into the endothelial cells. eNOS mRNA and protein expression were examined by RT-PCR and Western Blotting, respectively. Luciferase activity reporter system was used to study the target of 27nt-miRNA.
The results showed that overexpression of 27nt-miRNA significantly inhibited eNOS mRNA level and protein expression, and reduced the eNOS transcriptional efficiency. Such inhibitory effects of 27nt-miRNA were attenuated by the sequence mutations in 27nt-miRNA. Interestingly, the transcription factor SP-1 expression was reduced by 27nt-miRNA. Meanwhile, overxpression of SP-1 protein partially restored eNOS expression, and rescued the 27nt-miRNA-mediated reduction of endothelial cell proliferation. Moreover, certain sites in the SP-1 mRNA were found to be the direct target of 27nt-miRNA by a luciferase reporter system.
These results demonstrate that the 27nt-miRNA suppresses eNOS gene expression and SP-1 expression in vascular endothelial cells. The 27nt-miRNA directly target to SP-1 mRNA, thereby contributing to proliferation of endothelial cells.
本研究旨在探讨 27nt-miRNA 介导的内皮型一氧化氮合酶（eNOS）基因表达调控的分子机制。
通过将 miRNA 表达载体转染内皮细胞，建立过表达 27nt-miRNA 或其突变体的细胞系。通过 RT-PCR 和 Western Blotting 分别检测 eNOS mRNA 和蛋白表达。使用荧光素酶活性报告系统研究 27nt-miRNA 的靶标。
结果表明，过表达 27nt-miRNA 显著抑制 eNOS mRNA 水平和蛋白表达，并降低 eNOS 转录效率。27nt-miRNA 的这种抑制作用可通过 27nt-miRNA 序列突变减弱。有趣的是，SP-1 转录因子的表达被 27nt-miRNA 降低。同时，SP-1 蛋白的过表达部分恢复了 eNOS 表达，并挽救了 27nt-miRNA 介导的内皮细胞增殖减少。此外，通过荧光素酶报告系统发现 SP-1 mRNA 的某些位点是 27nt-miRNA 的直接靶标。
这些结果表明，27nt-miRNA 抑制血管内皮细胞中 eNOS 基因表达和 SP-1 表达。27nt-miRNA 直接靶向 SP-1 mRNA，从而促进内皮细胞的增殖。
