靶向调控血管生成素miRNA的筛选和验证

Screening and Functional Analysis of Angiogenin-targeting mi RNAs

血管生成素是一个重要的促血管生成因子,在细胞增殖、迁移和凋亡等过程中均发挥重要作用,但其具体的分子机制尚待阐明.mi RNA是一类长约22 nt的小RNA,在转录后水平调控基因的表达,广泛参与各种生物学过程.本文探索了可直接调控血管生成素表达的mi RNA,希望为阐明血管生成素的作用机制提供线索.首先,我们利用数据库预测得到8个可能靶向结合血管生成素m RNA 3'端非编码区的mi RNA;然后,用实验方法验证它们与血管生成素的靶向关系,发现mi R-1208、mi R-196b、mi R-296、mi R-409-3p、mi R-570和mi R-641这6个mi RNA可以不同程度地抑制血管生成素的m RNA和蛋白质表达水平,但只有mi R-196b、mi R-296、mi R-409-3p和mi R-641可以直接结合血管生成素m RNA的3'端非编码区;进而,在血管内皮细胞中分别过表达这4个mi RNA,发现mi R-196b、mi R-409-3p和mi R-641可以抑制血管内皮细胞的细胞增殖,而mi R-196b、mi R-296和mi R-409-3p可以抑制血管内皮细胞的管腔形成.以上结果表明,细胞内有多个mi RNA调控血管生成素的表达,它们可能协调调节血管生成,抑或在血管生成的不同阶段发挥作用.我们的工作还为"一种m RNA可被多种micro RNA调节,而一种micro RNA可调节多种m RNA"假说提供了部分证据.

Angiogenin （ANG） is a critical angiogenic factor that plays important roles in cell proliferation, migration and apoptosis. However, the mechanism of ANG-induced angiogenesis is still unclear. MicroRNAs （miRNA） are small non-coding RNAs of about 22 nt in length that regulates gene expression post-transcriptionally. Hence, miRNAs play important roles in many biological events, certainly includes angiogenesis. The miRNAs involved in ANG participated biological processes were largely unidentified. To find miRNAs targeting to ANG-3＇UTR, potential candidates were predicted by bioinformatics analysis, from which 8 miRNAs were selected for validation. Six miRNAs （miR-1208, miR-196b, miR-296, miR-409-3p, miR-570 and miR-641 ） were shown to inhibit ANG mRNA and protein expression, including 4 miRNAs （miR-196b, miR-296, miR409-3p and miR-641 ） directly targeted to ANG-3＇UTR. In HUVECs, cell proliferation was inhibited by overexpression of miR-296, miR-409-3p or miR-641; tube formation was repressed by miR-196b, miR-296 or miR409-3p overexpression. Our data indicated that four miRNAs were able to regulate ANG expression.