Notch通过Slit/miR-218/Robo1信号调控血管形成的作用和机制研究

During angiogenesis, Notch signaling regulates the formation and growth of sprouts by several mechanisms including the segregation of tip and stalk cells. However it has not yet been completely understood how Notch signaling regulates sprouting and results in differential phenotypes of endothelial cells. Recently, we compared miRNA expression profiles between endothelial cells (ECs) with forced Notch activation (conditional overexpression of Notch intracellular domain or NICD) and wild type ECs by using small RNA sequencing, and found that miR-218 was highly expressed in ECs with Notch activation (equivalent to stalk cells). It has been reported that in ECs, miR-218 represses Robo1, which promotes angiogenesis. We therefore speculate that Notch signaling likely regulates sprouting and the differentiation of tip and stalk cells through Slit/miR-218/Robo1 pathway. In the current project, we will ① elucidate the mechanisms of Notch signal-mediated modulation of Slit/Robo1 through miR-218 in ECs; ② reveal the role of Notch signaling in regulating ECs phenotypes through Slit/miR-218/Robo1; and ③ clarify the significance of Notch- Slit/miR-218/Robo1 pathway in physiological and pathological angiogenesis such as tumor neovascularization. These studies will further reveal the molecular network underlying angiogenesis, and provide potentially novel strategies for intervening with pathological neovascularization.

血管形成中，Notch信号通过决定顶端细胞和茎细胞分化等环节调控血管芽的生长，但Notch作用的下游分子和机制仍不完全清楚。最近，申请人用小RNA测序对比了来自条件激活性Notch胞内段转基因小鼠的内皮细胞和野生型内皮细胞的miRNA表达谱，发现在Notch激活的内皮细胞（相当于茎细胞）中，miR-218表达明显升高。miR-218抑制Robo1，而Robo1促进血管形成。因此Notch信号很可能通过miR-218抑制Robo1来调控血管形成中血管芽的生长。本课题拟①明确内皮细胞中Notch信号通过miR-218调控Robo1的机制；②阐明Notch-Slit/miR-218/Robo1信号对内皮细胞表型的调控作用；③揭示Notch-Slit/miR-218/Robo1信号对生理和病理性血管形成的作用和机制。这些研究不仅可进一步阐明血管形成的分子调控网络，还可为其干预提供潜在新策略。

血管形成（angiogenesis）是成体血管再生的基本形式，参与多种疾病的发生和进展。Notch信号调控内皮细胞（endothelial cell， EC）分化为顶端细胞和茎细胞，但Notch如何影响两种细胞的表型差异尚不明确。课题组前期发现在EC中Notch活化可以上调一组miRNAs，其中miR-218可在多种细胞中调控细胞增殖和迁移。因此，本研究探讨了Notch调控miR-218影响EC功能和血管形成的作用和机制，以及Notch-miR-218信号对病理性血管形成的调控作用和医学意义。. 结果发现，在EC中活化Notch可上调miR-218及其宿主基因Slit2的表达；而阻断Notch可下调miR-218及Slit2表达；Notch可激活Slit2启动子区报告基因。这些结果证实存在于Slit2基因第14内含子的miR-218是Notch的新型下游基因。向EC转染miR-218，可抑制EC增殖，造成管腔形成和出芽能力下降。利用RNA-seq发现miR-218过表达抑制MYC regulon，并得到qRT-PCR和Western blotting证实。在转染miR-218的同时过表达MYC，可逆转EC增殖抑制，证实miR-218通过MYC抑制EC增殖。进一步发现，miR-218可下调MYC的mRNA水平，还可靶向hnRNPA1和EYA3分别抑制MYC翻译以及降低MYC蛋白稳定性。在EC中转染NICD的同时抑制miR-218表达，可恢复EC增殖和出芽能力，说明Notch信号通过上调miR-218抑制MYC、EC增殖和血管出芽。最后，我们发现在新生小鼠，玻璃体注射miR-218 Agomir，可显著下调MYC蛋白水平，抑制EC增殖；在成年小鼠建立了脉络膜血管新生（CNV）模型，玻璃体注射miR-218 Agomir可显著降低CNV病理变化。这些结果初步证实miR-218可抑制CNV进展，可能是CNV相关疾病的治疗策略。此外，我们还探讨了其他Notch下游miRNAs在EC中的作用和机制，以及Notch信号对miRNAs通过细胞外囊泡的旁分泌和对正常和肿瘤干细胞的调控作用。.已发表相关研究论文10篇。授权发明专利1项，公开1项，先后有2位博士研究生、5位硕士研究生、1位博士后参与科研工作。主编国家卫生健康委员会“十三五”规划教材《医学分子生物学实验技术》1部。