血管生成素靶向调控miR-141的分子机制

Angiogenin (ANG) plays key roles in tumor progression and neurodegenerative diseases, but the underlying mechanisms remain unclear. Recent studies have shown that, as a member of RNase super family, the ribonucleolytic activity of ANG is crucial for its biological functions, e.g., by processing rRNA and tRNA. As a new family of RNA, microRNAs (miRNAs) are regulators of many biological events, including tumorigenesis, development, etc, by post-transcriptional inhibition on protein translation or decay of mRNA. Our recent studies have found that ANG down-regulates expressions of a number of miRNAs in human umbilical vein endothelial cells (HUVECs). Among them, miR-141, the most down-regulated one, can inhibit angiogenesis; furthermore, our data shows that ANG regulates angiogenesis through down-regulating miR-141. We have found that ANG does not affect the expression level of the primary miR-141 gene transcript, pri-miR-141, indicating that ANG regulates miR-141 expression through post-transcriptional pathways. Interestingly, the ANG mutant with mutation of key amino acid in ribonuclease domain (K40I) can not down-regulate miR-141 expression. In addition our in vitro study has shown that treatment of total RNA with ANG can decrease the amount of miR-141 copies. These data drive us to hypothesize that ANG down-regulates miR-141 expression through miR-141 degradation by ribonucleolytic activity. Thus, in this study, we will 1) confirm the role of ribonucleolytic activity of ANG in regulation of miR-141 expression in HUVECs; 2) identify the processing sites of miR-141 during its degradation by ANG. Our study will elucidate the molecular mechanisms of ANG in regulating miR-141 biogenesis. If we could determine that miRNA is a substrate of ANG, it would be an innovative finding.

血管生成素（angiogenin, ANG）在肿瘤和神经退行性疾病中发挥重要作用，但其分子机制尚未完全阐明。已知ANG的核糖核酸酶活性在其发挥功能时是必需的，并已鉴定了rRNA和tRNA两类底物。申请人的前期研究发现，ANG可下调一批miRNAs的表达，且对miR-141的下调作用与核糖核酸酶活性密切相关。为此，本项目拟利用ANG的酶活性缺失突变体和专一性抑制剂等，首先在细胞水平明确核糖核酸酶活性在ANG下调miR-141表达中的作用和发挥作用的方式；然后，利用酶学和分子生物学等技术，确定ANG直接作用的底物及其剪切位点。通过本项目研究，不仅将阐明ANG下调miR-141表达的分子机制，而且可能发现ANG下调miRNA表达的普遍机制。总体上，证明miRNA是ANG的底物将是一项创新性的发现。

成熟miRNA降解是miRNA生物学过程的一个重要步骤，目前尚未报道核糖核酸内切酶在该过程中发挥作用。血管生成素（angiogenin，ANG）在肿瘤和神经退行性疾病中发挥重要作用，但其分子机制尚未完全阐明。ANG是核糖核酸酶超家族的成员，其核糖核酸酶活性很微弱但是其发挥生物学功能的必需，rRNA和tRNA是ANG的两类底物，但ANG是否对miRNA具有酶解作用尚未见报道。前期研究发现ANG下调miR-141表达且与其核糖核酸酶活性密切相关。经本项目的研究，我们发现ANG在转录后水平特异性下调成熟miR-141，而不影响pre-miR-141和pri-miR-141的水平，也不影响具有相似序列的miR-200家族其他成员的水平。而且ANG对miR-141的调控作用依赖于ANG的核糖核酸酶活性，当ANG的核糖核酸酶活性缺失或被抑制时，ANG对miR-141的调控功能消失。同时，我们发现ANG与miR-141在细胞内相互结合并在细胞质中共定位，ANG对miR-141在细胞内的降解作用也得到验证。我们通过体外剪切系统进一步分析ANG对miR-141的剪切形式和剪切位点，发现ANG以核糖核酸内切酶的形式剪切miR-141序列中的5A、7U、11U和14U四个位点，而且1分子ANG在30分钟内降解5个分子的miR-141。ANG只对单链的miR-141进行剪切，对DNA序列、双链形式的miR-141均没有剪切作用，其中核苷酸的甲基化修饰可以阻断ANG对miR-141的剪切作用，但尿苷化和腺苷化修饰则不影响。当miR-141处于二级结构状态时，ANG只对其中处于单链环状结构位置的14U进行剪切，对处于或靠近双链结构的5A、7U和11U位点则没有剪切作用。此外，我们发现在细胞内ANG对miR-141的剪切位点与体外剪切有差异。最后，我们探索了ANG对其他miRNA的剪切机制以及验证了ANG调控miR-141及靶基因这个调控通路在血管生成和结直肠癌发生发展中的作用。.综上所述，我们发现了miRNA在哺乳动物细胞中降解的新机制，阐明了ANG下调miR-141的分子机制，证明miRNA是ANG酶解的一类新的底物以及ANG是迄今第一个降解成熟miRNA的核糖核酸内切酶，并验证该调控机制在血管生成和结直肠癌发生发展中的重要作用，该成果具有原始创新性并为结直肠癌的诊断和治疗提供新的靶标。