长链非编码RNA WTAPP1对内皮祖细胞功能的调节机制及其在深静脉血栓中转化应用的实验研究

Endothelial progenitor cell (EPC) plays an important role in the recanalization of deep vein thrombosis (DVT). But there are a few numbers of EPC in peripheral blood. Furthermore, EPC and its function are also confined by age and other risk factors, which will impair the roles of recanalizaion in DVT. Thus it is very important to improve the capacities of homing and angiogenesis. According to our previous study, we found that the expression of lncRNA WTAPP1 in EPC from DVT patients was lower than that from healthy donors. Metal Matrix Proteinase 1 (MMP1) was identified as the target gene of lncRNA WTAPP1. Besides, miR-9-5p also can target on MMP1 and expressed higher in the cells with lncRNA WTAPP1 siRNA. MMP1 plays an important role in the migration and angiogenesis. Thus, we suspected that lncRNA WTAPP1 could promote MMP1 expression through the regulation of MiR-9-5p, and further enhance the migration and angiogenesis of EPC. In this study, ①. We will find out the role of lncRNA WTAPP1 in homing and angiogenesis of EPC; ② identify the mechanisms of the regulation of MMP1 by lncRNA WTAPP1 through miR-9-5P; ③. Explore the role of lncRNA WTAPP1 in the assessment of DVT prognosis via animal model of thrombosis. This project will provide an important theoretical reference for stem cell treatment and prognosis evaluation of DVT.

内皮祖细胞（EPC）在深静脉血栓形成（DVT）的再通中起着重要作用，但外周血中EPC含量较少且易受年龄等因素的影响，使其作用效果减弱，如何提高其归巢和成血管能力显得尤为重要。我们前期发现DVT患者EPC中lnc-WTAPP1表达降低，此lncRNA不仅与对细胞归巢和血管新生起重要作用的金属基质蛋白酶1（MMP1）在遗传学上相关，还与miR-9的表达呈负相关，同时后者也与MMP1相关。据此推测lnc-WTAPP1可能通过影响miR-9来调控MMP1表达进而提高EPC在血栓再通中的作用。本研究将①探明lnc-WTAPP1对EPC归巢及成血管能力的影响；②研究lnc-WTAPP1通过miR-9来调控MMP1的分子机制；③通过动物血栓模型解析EPC参与调控DVT转归过程中lnc-WTAAP1扮演的角色。该项目的完成将提出EPC归巢及成血管的新机制及为DVT的干细胞治疗及转归评价提供重要的理论依据。

内皮祖细胞（EPC）在深静脉血栓形成（DVT）的再通中起着重要作用，但外周血中EPC含量较少且易受年龄等因素的影响，使其作用效果减弱，如何提高其归巢和成血管能力显得尤为重要。本项目已完成相关研究：（1）lnc-WTAPP1可调控EPC迁移、侵袭和成血管能力：下调EPC中WTAPP1表达会抑制细胞迁移、增殖和成血管功能；反之。（2）lnc-WTAPP1 能调控EPC中MMP-1表达：改变EPCs中lnc-WTAPP1表达后，发现其与MMP-1存在正相关调节关系。（3）Lnc-WTAPP1通过PI3K/Akt/mTOR 信号通路调控EPCs 生物功能：敲减Lnc-WTAPP1，EPC中PI3K, Akt表达下调，mTOR磷酸化水平降低；反之。（4）Lnc-WTAPP1作为miR-3120-5p 诱饵在外周血来源的EPCs中发挥调控作用：过表达 lncRNA WTAPP1的 EPCs 中miR-3120-5p表达降低，二者存在负相关关系。miR-3120-5p通过MMP1 的 3' UTR端结合从而抑制其表达。（5）Lnc-WTAPP1通过调节自噬过程影响EPCs细胞功能：改变EPCs中Lnc-WTAPP1表达后，检测到细胞中自噬相关基因LC3, Atg5和P62表达也随之发生显著性改变。（6）miR-3120-5P 抑制EPC迁移和成血管功能。以上研究结果明确了 lncRNA WTAPP1 的临床意义，为干细胞治疗 DVT 的临床前研究及临床试验提供理论依据。