长链非编码RNA（lncRNA）——GAS5对人大隐静脉曲张调控的机制研究

Long non-coding RNAs (lncRNAs) are a class of transcripts with more than 200 nt length but without protein-coding potentiality. As key regulatory molecules, lncRNA widely functioned in the regulation of cardiovascular diseases, however, the roles of lncRNAs play in the development of the primary varicose great saphenous veins (PVGSVs) remain unclear. As the most common chronic venous disease,the PVGSVs’ pathogenesis is not yet fully clear but one of the most important mechanisms is the imbalance between the proliferation and the apoptosis of the vessel wall cells. Based on our previous work, lncRNA-GAS5 was identified aberrantly expressed in varicose segments of PVGSVs by Q-RT-PCR. The human saphenous vein smooth muscle cells (HSVSMCs) were isolated and cultured for the functional study of GAS5. The HSVSMCs were transfected siRNA and overexpression plasmid of GAS5 to interfere its expression level and then HSVSMCs proliferation, migration, apoptosis and cell cycle were detected changed. Annexin A2 was identified a direct binding protein to GAS5 using RNA Pulldown assay and MALDI-TOF-MS. Both GAS5 and Annexin A2 were proved to play a key role in regulation of mammalian cells proliferation and apoptosis process. Therefore, we hypothesized that GAS5 may regulate the proliferation and apoptosis function of vessel wall cells through Annexin A2, and eventually participate in the pathogenesis of PVGSVs even more vascular hyperplastic disease. This project intends to build the vascular smooth muscle cell and endothelial cell models of great saphenous vein. Then we will intervene the expression of GAS5 and detect the changes of cell proliferation, apoptosis, migration, cycle, and other functional phenotypes even more the related signaling pathways in the cell models. After then, we will explore whether GAS5 function through Annexin A2 and its possible mechanisms from the cellular and molecular levels. In this study, we aim to provide novel insights into physiology of lncRNAs and pathophysiological properties of PVGSVs, meanwhile, serve as new therapeutic targets for varicose veins.

长链非编码RNA（lncRNA）是一类转录本长度超过200nt的不具有编码蛋白质功能的RNA，广泛参与心血管疾病调节。血管壁细胞增殖凋亡失衡是大隐静脉曲张的重要发病机制。我们前期工作筛选出lncRNA-GAS5在曲张大隐静脉中显著下调，证实干预GAS5可引起大隐静脉血管平滑肌细胞增殖、凋亡、迁移、周期变化，并鉴定出GAS5的直接结合蛋白Annexin A2。GAS5和Annexin A2均被发现对细胞的增殖和凋亡起关键调控作用。推论：GAS5可能通过影响Annexin A2调控血管壁细胞的增殖和凋亡，最终参与大隐静脉曲张发病。本项目拟建立大隐静脉血管平滑肌和内皮细胞模型，干预GAS5并检测细胞增殖、凋亡、迁移、周期等功能表型及相关信号通路变化，并探索GAS5是否通过Annexin A2发挥作用及其可能机制，以期丰富lncRNA的理论知识，为大隐静脉曲张的诊治提供新的分子标志和药物靶点。

长链非编码RNA（lncRNA）是一类转录本长度超过200nt的不具有编码蛋白质功能的RNA，广泛参与心血管疾病调节。血管壁细胞增殖凋亡失衡是大隐静脉曲张的重要发病机制。我们前期工作筛选出lncRNA-GAS5在曲张大隐静脉中显著下调，证实干预GAS5可引起大隐静脉血管平滑肌细胞增殖、凋亡、迁移、周期变化，并鉴定出GAS5的直接结合蛋白Annexin A2。GAS5和Annexin A2均被发现对细胞的增殖和凋亡起关键调控作用。推论：GAS5可能通过影响Annexin A2调控血管壁细胞的增殖和凋亡，最终参与大隐静脉曲张发病。在本研究中，我们收集了人大隐静脉曲张血管标本，成功的进行人大隐静脉血管平滑肌细胞（HSVSMCs）及内皮细胞（HSVECs）的分离培养鉴定，并在这些细胞模型上对GAS5的表达量进行干预（敲低：siRNA、shRNA RNAi/过表达：构建过表达质粒，使用慢病毒载体建立稳转株），对细胞各种生物学活性（如增殖、凋亡、迁移、细胞周期等）进行检测。结果证实：在 HSVSMCs 内敲低 GAS5，促进细胞增殖，迁移加速，加快细胞周期进程，但并不显著影响细胞的凋亡和坏死。反之，过表达 GAS5，细胞增殖抑制，周期进程减慢，但凋亡减轻。而在HSVECs中，过表达GAS5，内皮细胞凋亡明显增加，但是并不影响细胞周期；而敲低GAS5，内皮细胞凋亡变化不明显，但是细胞周期变慢。根据我们前期工作，通过RNA Pulldown及质谱分析，筛选出Annexin A2是GAS5的结合蛋白。在本研究中，我们在HSVSMCs上，对GAS5、Annexin A2的表达分别进行干预，发现分别干预GAS5、Annexin A2并不影响Annexin A2、P11的表达量变化，证明GAS5与Annexin A2之间不存在单纯的上下游通路关系；此外，对GAS5、Annexin A2同时进行干预，发现敲低Annexin A2会抑制敲低GAS5引起的细胞增殖作用，预示Annexin A2参与GAS5对HSVSMCs生物活性功能的调节。后续我们将从细胞及分子层面继续研究GAS5是否通过影响Annexin A2的活性、定位或形成复合体来发挥功能及其具体分子机制，以期通过本课题的研究，既丰富lncRNA生物学功能和作用机制的理论知识，又为原发性大隐静脉曲张的诊断和治疗提供新的分子标志和药物靶点。