剪切应力抑制GAS5内源性miR-21“海绵”效应对EPCs修复损伤内皮的影响及机制

Vascular endothelial injury is associated with the occurrence and progression of many cardiovascular diseases，such as atherosclerosis, coronary artery disease and hypertension. Re-endothelialization is critical to restoration of normal vascular homeostasis and regulation of neointimal hyperplasia. The endothelial progenitor cells (EPCs) recently emerged as an important component of the response to vascular injury, having the potential to accelerate vascular repair through rapid re-endothelialization. Previous studies have revealed shear stress could accelerate EPC proliferation, differentiation, migration and so on, which contribute to vascular repair in vivo.However, the underlying mechanisms of shear stress promoting EPC functions have not been understood completely. Long non-coding RNAs (LncRNAs), often referred to as the “Dark Matter” of the genome, are emerging as an integral functional component of the mammalian transcriptome. Although our understanding of LncRNAs is still in its infancy in the cardiovascular field, increasing evidence suggests that LncRNAs play important roles in the regulation of tissue homeostasis and pathophysiological conditions. To explore the role of LncRNAs in EPC functions regulated by shear stress, we analyzed the expression profiles of LncRNAs, in 3 paired static control and shear stress treated EPCs, using a microarray. The results of microarray analysis indicated a significant difference in LncRNA expression between static control and shear stress treated EPCs. A total of 28 LncRNAs (fold change ≥4.5, P value <0.05) were found to be differently expressed in shear stress treated EPCs compared to the static controls. Among these, LncRNA GAS5 was the most significantly down-regulated. Based on our preliminary data and bioinformatics analysis, here we put forward the hypothesis: shear stress-sensitive GAS5 functions as an endogenous miR-21 “sponge” to regulate the expression of miR-21 target gene, PTEN. Namely, LncRNA GAS5 acts as a ceRNA, and formed a feedback loop with miR-21 and PTEN in shear stress treated EPCs. Furthermore, GAS5-miR-21-PTEN mRNA regulatory loop can modulate functions of EPCs in response to shear stress through activating the PI3K/Akt-eNOS pathway. To test the hypothesis, we will apply multidisciplinary techniques, such as real time RT-PCR, Western blot, luciferase assay, RNA immunoprecipitation, cell transplantation and so on. These studies would have a high probability of revealing the mechanism of vascular endothelial repair and providing new directions that lead to the development of clinical strategies toward the prevention and/or treatment of cardiovascular diseases induced by vascular endothelial injury.

血管内皮损伤与心血管疾病发生发展密切相关，增强内皮祖细胞（EPCs）功能活性可促进损伤血管的再内皮化。国内外资料和我们研究均表明：剪切应力可调控EPCs的功能活性，但其机制有待不断完善。长链非编码RNAs(LncRNAs)长期以来被视为基因组转录的“暗物质”，目前LncRNAs在心血管领域的研究刚刚起步。本课题组根据前期LncRNAs芯片结果、预实验及生物信息学分析提出：在剪切应力作用下，EPCs内具有内源性miR-21“海绵”效应的LncRNA GAS5表达下调，对miR-21的竞争性抑制作用减弱，加速了miR-21对其靶基因PTEN的降解，引发Akt激活、eNOS表达上调等信号事件，提升了EPCs修复损伤内皮的能力。本研究将从分子、细胞、整体层面对上述假设进行求证，以全新的视角阐明剪切应力调控EPCs功能活性的分子机制，为临床开展剪切应力介导的细胞疗法提供新的作用靶点和实验依据。

血管内皮损伤与心血管疾病发生发展密切相关，增强内皮祖细胞（EPCs）功能活性可促进损伤血管的再内皮化。国内、外资料和我们前期研究均表明：剪切应力可调控EPCs的功能活性，但其机制尚不明晰。目前LncRNAs在心血管领域的研究已成为热点。本研究从分子、细胞、整体层面探讨剪切应力对LncRNAs的调控；LncRNAs——GAS5对EPCs功能影响及机制等科学问题。结果显示：（1）剪切应力（12 dyne/cm2, 12 h）可改变EPCs LncRNAs表达谱。剪切应力作用后，共有742条LncRNAs显现为差异表达，其中表达差异4.5倍以上的有28条，以GAS5的降低最为显著。（2）GAS5可靶向结合miR-21，进而降低miR-21与PTEN 3’UTR相应位点的结合而发挥对PTEN的转录后调控，即在EPCs内存在GAS5 ceRNA（GAS5- miR-21-PTEN mRNA）调控模式。（3）GAS5 ceRNA系统内GAS5、 miR-21及PTEN分子具有剪切应力响应性，剪切应力下调GAS5、PTEN的表达，但明显促进miR-21的表达。（4）剪切应力可上调EPCs的分化、增殖、粘附等能力。重构GAS5 ceRNA（GAS5-miR-21-PTEN mRNA）系统，对剪切应力促EPCs功能有一定调控作用，LV-GAS5、miR-21 inhibitor及LV-PTEN均不同程度地抑制剪切应力促EPCs功能的效应。（5）剪切应力可激活Akt，增加eNOS表达，促进NO生成。LV-GAS5、miR-21 inhibitor及LV-PTEN均不同程度抑制了剪切应力激活Akt、促eNOS表达及NO生成的效应，提示Akt-eNOS通路是GAS5 ceRNA系统的下游信号。(6) PI3K/Akt 激动剂740 Y-P、NO供体SNP，可不同程度逆转GAS5 ceRNA系统对剪切应力促EPCs功能的影响。以上结果，以全新的视角阐明了剪切应力调控EPCs功能活性的分子机制，完善了受损血管内皮修复的力学、生物学机制，为动脉粥样硬化等心血管疾的发病机理及剪切介导的细胞疗法提供理论依据。