CircRNA_0006799竞争性结合miR-1269a调控PTEN影响下肢动脉硬化闭塞症血管平滑肌细胞生物学行为的机制研究

Atherosclerosis is a disease of the cardiovascular system that is characterized by chronic inflammation of the arterial wall. The regulation and function of artery smooth muscle cells (ASMCs) are important in the progression of atherosclerosis. In atherosclerosis, excessive proliferation and migration of ASMCs play a key role in the remodelling of arterial wall. Circular RNA(circRNA) is a kind of endogenous RNA which plays an important role in transcriptional regulation. Its specific effect in the development of atherosclerosis is still unknown. CircRNA associates with related miRNA and the circRNA-miRNA axes are involved in a series of disease pathways such as apoptosis, vascularization, invasion and metastasis.. In our previous study, we have identified the gene expression profiles of circRNA, miRNA and mRNA of Arteriosclerosis Obliterans specimen and normal lower limb artery specimen using RNA sequencing (RNA-seq) technologies and bioinformatics. The results show that circ_0006799 is significantly downregulated, miR-1269a is significantly upregulated, and phosphatase and tensin homology deleted on chromosome ten (PTEN) mRNA is significantly downregulated in the Arteriosclerosis Obliterans specimen compared with normal lower limb artery specimen. But the biological function of circ_0006799 in the pathogenesis of Arteriosclerosis Obliterans and its specific molecular mechanism are still unknown.. Further study shows that silencing of circ_0006799 with siRNA significantly stimulates the proliferation and migration of ASMCs, upregulates the level of miR-1269a, and downregulates the expression of PTEN. PTEN is a famous tumor suppressor which is expressed in many kinds of tissues as a constitutive secretion protein. PTEN is known to be involved in the regulation of a variety of physiological and pathological processes through negative regulation of the PI3K/AKT signaling pathway. Bioinformatics analysis shows that circ_0006799 is potentially a sponge for miR-1269a, which can target PTEN. . Based on these studies, we put forward a scientific hypothesis that circ_0006799 could promote the proliferation and migration of ASMCs by targeting miR-1269a /PTEN, then activating the PI3K/AKT signaling pathway. The results of this study will explore the vital role of circular RNA in the pathogenesis and development of Arteriosclerosis Obliterans, and provide theoretical supports for the potential new treatment methods.

动脉平滑肌细胞（ASMCs）增殖和迁移异常是下肢动脉硬化闭塞症（AS0）发病的主要原因。环状RNA（circRNA）在其中的作用尚不清楚。前期研究表明，下肢ASO组织中circ_0006799下调，miR-1269a上调，PTEN mRNA下调。进一步预实验发现，去表达circ_0006799可以诱导ASMCs增殖和迁移，上调miR-1269a，下调PTEN的表达。分析显示，circ_0006799与miR-1269a具有结合靶点，而PTEN是miR-1269a的靶基因。PTEN可以调控多种生物学进程。据此我们提出科学假说：circ_0006799通过竞争性结合miR-1269a下调PTEN表达，促进ASMCs增殖迁移，抑制凋亡。本项目拟采用分子生物学实验结合动物模型的方法，阐明circ_0006799在ASMCs异常生物学行为中的作用及其具体分子机制，为寻找ASO治疗新靶点提供科学依据。

下肢动脉硬化闭塞症（Arteriosclerosis Obliterans，ASO），是全身动脉粥样硬化性疾病的局部表现，其确切的发病机制不明确。其中，动脉平滑肌细胞（ASMCs）增殖和迁移异常是下肢动脉硬化闭塞症（AS0）发病的主要原因。circRNA是近年来RNA研究领域的热点, circRNA在下肢ASO组织中的表达谱以及在发病过程中的生物学功能和具体的分子机制仍未见报道。考虑到circRNA可能在下肢ASO的发病中扮演重要角色,我们进行了本课题的研究。.本研究的主要内容是通过一系列细胞实验和动物实验，证实circ_0006799的生物学功能以及具体的作用机制。具体内容包括：1、观察circ_0006799对ASMCs增殖、迁移、凋亡的影响：在细胞水平检测升高或者降低circ_0006799水平后ASMCs增殖、迁移、凋亡的变化；2、分析circ_0006799影响ASMCs功能是通过竞争性抑制miR-1269a下调PTEN，减弱对PI3K/AKT通路的抑制而实现；3、构建慢病毒过表达circ_0006799载体，转染至大鼠颈动脉球囊损伤模型，分析circ_0006799体内抑制动脉狭窄的能力.本项目中，我们对下肢ASO病变动脉标本及正常下肢动脉标本进行circRNA高通量芯片筛查。结果发现，共有12000多种circRNA在人体股动脉标本中表达。通过原位杂交免疫荧光双染色技术，我们对ASO组织和正常动脉壁进行分析，发现circ_0006799主要表达在动脉中层的血管平滑肌细胞上，且病变组织的表达量较正常组织明显下降。构建circ_0006799过表达及去表达慢病毒载体后，我们通过对ASMCs和ASO动物模型的一系列实验，证明circ_0006799是通过PTEN蛋白，减弱对PI3K/AKT通路的影响，而发挥作用。.经过研究，我们得出结论：circ_0006799在下肢ASO发病中通过调控ASMCs增殖、迁移、凋亡发挥作用，具体机制是：通过竞争性抑制miR-1269a，下调PTEN的表达，减弱对PI3K/AKT通路的抑制。.本研究的成功，揭示了circ_0006799在下肢ASO发病过程中的生物学功能及其具体的分子机制。研究结果将为下肢ASO的诊断和治疗提供新的理论依据。