LncRNA-ITGA1-1上调整合素α2表达促进动脉粥样硬化病变中血管平滑肌细胞增殖的机制研究

Aberrant vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to the development of vascular pathologies, such as atherosclerosis and restenosis. Long noncoding RNAs (LncRNAs) have emerged as important regulators for VSMC function, however, the molecular mechanisms still remain unclear. We recently identified lnc-ITGA1-1 as critical for controlling human aortic smooth muscle cell (hAoSMCs) proliferation and migration. By using quantitative reverse-transcription polymerase chain reaction, we found that lnc-ITGA1-1 was significantly downregulated in hAoSMCs on platelet-derived growth factor (PDGF) treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that knock down of lnc-ITGA1-1 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited PDGF-induced VSMC proliferation and migration. Therefore, the aim of this study is to clarify the role of lnc-ITGA1-1 in regulating integrin α2（ITGA2）in nucleus and cytoplasm. On the one hand, ChIRP and ChIP were used to explore the epigenetic modifications of promoter region of lnc-ITGA1-1 in the nucleus; on the other hand, combined with RNA-pull down and Deletion Mapping, the aim of this study was to explore the effects of lnc-ITGA1-1 in cytoplasm on the stability and downstream signal pathway activity of ITGA2 protein. Overall, the mechanism of lnc-ITGA1-1 in VSMC proliferation and migration will be systematically verified in the present study, which provides a new evidence for the targeted treatment of atherosclerosis (AS).

血管平滑肌细胞（VSMC）过度增殖是动脉粥样硬化（AS）的主要致病因素，lncRNA在其中的调控机制尚未阐明。本课题组前期发现，lnc-ITGA1-1在增殖的人VSMCs和AS组织中表达升高，敲减lnc-ITGA1-1后可抑制VSMC增殖和迁移，同时抑制了整合素α2（ITGA2）的表达水平及其下游信号的活化。因此，本课题拟针对lnc-ITGA1-1在转录及转录后水平调控ITGA2的表达展开研究。运用ChIRP和ChIP等手段明确胞核lnc-ITGA1-1对ITGA2启动子区表观修饰及基因转录活性的调控作用；利用RNA-pull down和Deletion Mapping等技术阐明胞质内lnc-ITGA1-1与ITGA2蛋白的结合位点、检测其对ITGA2蛋白稳定性以及对下游信号通路活性的影响；从而系统探索lnc-ITGA1-1对VSMC过度增殖和迁移的调控机制，为AS的靶向治疗提供新思路。

血管平滑肌细胞（VSMC）过度增殖是动脉粥样硬化（AS）的主要致病因素，lncRNA在其中的调控机制尚未阐明。本课题发现，lnc-ITGA1-1在增殖的人VSMCs和AS组织中表达升高，敲减lnc-ITGA1-1后可抑制VSMC增殖和迁移，同时抑制了整合素α2（ITGA2）的表达水平及其下游信号的活化。LncRNA-ITGA-1来源于整合素α2（ITGA2）基因内含子区域，参与PDGF-BB诱导的VSMC增殖和迁移。基于生物信息学分析和荧光素酶分析，发现lncRNA-ITGA-1的互补DNA序列位于增强子内。芯片和ChIRP分析显示，lncRNA-ITGA-1通过其互补链直接与增强子结合，并增加该区域以及ITGA2启动子的H3K27乙酰化。此外，lncRNA-ITGA-1可以与DNA结合蛋白NONO相互作用，后者同时与ITGA2启动子结合。值得注意的是，与非冠心病患者相比，冠心病患者的血浆和冠脉组织中lncRNA-ITGA-1显著上调。本研究发现了一种新的调控机制，其中lncRNA-ITGA-1以NONO依赖性方式介导其互补增强子区和ITGA2启动子之间的相互作用，以增加ITGA2的表达，从而促进VSMC增殖和迁移。这些发现为人类增殖性血管疾病的诊断和治疗提供了一个有吸引力的靶点。