切应力敏感lncRNA调控血管内皮炎症的分子机制研究

By regulating the gene expression of vascular endothelial cells (ECs), the physiological flow shear stress plays a major role in maintaining vascular endothelium homeostasis, subsequently inhibiting the development of atherosclerosis (AS). AS is initiated by inflammatory processes in the endothelial cells of the vessel wall. Inflammatory processes are important targets of therapy in preventing or treating AS. Our preliminary studies show that microRNAs modulate flow-dependent regulation of endothelial inflammation. However, it remains unclear that whether long non-coding RNAs (lncRNAs) are involved in flow-mediated regulation of endothelial inflammation. Our lncRNA microarray data show that, LINC00520 is upregulated by anti-inflammatory HSS (high shear stress) but downregulated by pro-inflammatory LSS (low shear stress). Subsequent investigation demonstrates that anti-inflammatory transcription factor KLF2 can upregulate the expression of LINC00520. Blockade of endogenous LINC00520 significantly promotes the expression of ECs inflammatory markers. The results suggested that LINC00520 might be involved in the flow-dependent regulation of endothelial inflammation. By using microarray-based gene expression profiling analysis and CRISPR system for gene regulation, the present study is conducted to further investigate whether mechanosensitive lncRNAs (including LINC00520) can modulate endothelial inflammation, to elucidate their molecular mechanisms and analysis their impact on progression of AS. The results will provide new theoretical guidance for the prevention and treatment of AS.

生理血流切应力通过调节血管内皮细胞（ECs）基因的表达，维持内皮结构与功能稳定，抑制动脉粥样硬化（AS）。内皮炎症是AS发生与防治的关键环节，申请者已阐明了多个切应力敏感microRNA对ECs炎症的调控作用，但目前尚无切应力敏感lncRNA与ECs炎症关系的报道。我们的lncRNA芯片结果表明，抗炎的生理切应力诱导LINC00520多个转录本的表达发生显著上调，而促炎的低切应力则抑制LINC00520的表达。进一步研究发现抗炎的转录因子KLF2可诱导LINC00520的表达，而沉默LINC00520则显著促进ECs炎症标志分子的表达。上述结果提示：LINC00520可能参与切应力对ECs炎症的调控。本项目拟运用表达谱芯片及CRISPR基因表达调控等技术，深入探讨LINC00520等切应力敏感lncRNA对ECs炎症及AS的调控作用，明确其分子机制，从而为防治AS提供新的理论依据。

血流切应力是指血流对血管内皮细胞（ECs）所施加的平行于血管壁的摩擦力，ECs是切应力的直接作用对象。当前的研究表明，血流动力学因素在动脉粥样硬化的发生发展过程中起关键作用，但其分子机制尚未完全明确，特别是有关长链非编码RNA（lncRNA）是否介导切应力对内皮炎症的调控作用仍不完全清楚。我们的lncRNA芯片结果表明，抗炎的生理切应力诱导LINC00520多个转录本的表达发生显著上调，而促炎的低切应力则抑制LINC00520的表达。进一步研究发现抗炎的转录因子KLF2可诱导LINC00520的表达，而沉默LINC00520则显著促进ECs炎症标志分子的表达。上述结果证明生理切应力诱导的LINC00520调控内皮炎症。我们同时使用高通量测序的方法筛选了受KLF2调控的lncRNA，获得了多个受切应力和KLF2双调控的lncRNA，并深入探讨了这些lncRNA（包括LINC01013、LINC00152等）的血管内皮炎症调控功能，评估了这些lncRNA的核质分布情况，分析了切应力和KLF2调控这些lncRNA的潜在机制。我们同时还探讨了CRISPR/dCas9技术在在血管内皮细胞中激活lncRNA表达的效率。在动物实验方面，我们建立了体内血管平滑区和弯曲处血管内皮的enface染色技术，用于炎症相关基因的在体检测；同时构建了异常切应力诱导的动脉粥样硬化APOE-/-小鼠模型。上述研究进一步揭示了血流切应力、lncRNA以及内皮细胞炎症三者间的相互关系，可为动脉粥样硬化的预防和治疗提供部分新的理论依据。