MicroRNA-352和microRNA-347在大鼠后肢高血流切应力诱导的侧支血管生长中的作用及机制研究

Fluid shear stress (FSS) is widely accepted as the principal mechanism responscible for collateral vessel growth. After an arterial occlusion, increased FSS initiates the growth of collaterals, but it restores only 35-40% of the maximal conductance of the replaced artery. However, if fluid shear stress is elevated, a remarkable growth of collateral vessels will occur, leading to the complete restoration of blod supply, even over the maximal collateral conductance of the arterial bed before occlusion. Therefore, an increased FSS is a key factor regulating collateral vessel growth. Recently, several studies have been done in elucidating how the elevated fluid stress promotes collateral vessel growth, however, the underlying molecular mechanism remains unclear. MicroRNAs (miRNAs) are a novel class of regulatory noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to 3' untranslated regions of target mRNAs. An increasing body of evidence indicates that miRNAs play an important roles in regulating cellular functions such as differentiation, proliferation, and migration. Moreover, a number of specific miRNAs have been demonstrated to be important contributors for angiogenesis. Therefore, miRNAs most likely represent a good means for invetigating the mechanism by which elevated FSS intensivly promotes collateral vessel growth. Most recently using miRNA arrays, we explored the miRNA expression profile in collateral vessels induced by elevated FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein in rat hind limbs. We found 91 differential miRNAs (＞2 fold changes) in this model as compared to the simply ligation of femoral artery. By real time PCR we verified several down-regulated miRNAs whose functions are unknown currently, for example miR-352 and miR-347. Morever, by in situ hybridization miR-352 and miR-347 were shown located at endothelial and smooth muscle cells. Furthermore, the bioinformatic algorithms analysis showed that the target genes of miR-352 and miR-347 involve cell proliferation and important signal pathways. Based on our preliminary experiment data, this project is designed to further investigate the role and mechanism of miRNAs, especially miR-352 and miR-347 in collateral vessels induced by elevated FSS using molecular biology, bioinformatic algorithms, immunocofocal microscopy and cell culture techniques. It is highly expected that the outcome of this project will add valuable information to our knowledge in understanding of the role of lasting increased FSS in collateral vessel growth and some specific miRNAs regulating collateral vessel growth will be uncovered. Thus, this project is of great importance both in basic and clinical research.

动脉堵塞后侧支血管在增加的血流切应力作用下适应性生长，但只能部分地恢复组织的血流。然而，如果提高切应力，则侧支血管的生长显著增加，血流完全恢复甚至超过正常水平。遗憾的是目前其机制仍不十分清楚。我们近来对从大鼠股动脉结扎并行结扎远侧端和股静脉吻合的高血流切应力模型分离出的侧支血管进行了miRNA芯片分析，发现和单纯股动脉结扎相比，差异2倍以上的miRNA有91种。我们用qRT-PCR证实了miR-352和miR-347等几种表达下调但功能不明的miRNA的表达。荧光原位杂交显示这两种miRNA定位在内皮细胞和平滑肌细胞。生物信息软件预测结果提示这两种miRNA的靶基因与血管生成和重要的生长信号通路有关。本项目拟在前期研究的基础上，深入研究在高血流切应力的侧支血管生长模型miR-352/miR-347对侧支血管生长的作用及机制。本项目的成功对侧支血管生长理论的发展和临床应用都具有重要价值。

本项目进行了如下研究：1. 研究了提高血流切应力诱导的大鼠后肢侧支血管microRNA（miRNA）表达谱变化；2. 研究了miR-352对培养的大鼠血管内皮细胞增殖、迁移、成管形等的作用以及对大鼠后肢侧支血管生长的影响；3. 研究了miR-352-IGF2R信号通路对内皮细胞和侧支血管自噬功能的调节作用；4. 研究了miR-497-5p在侧支血管的表达及miR-497-5p-IGF2R信号通路对SD大鼠血管内皮细胞增殖、迁移、成管形等的作用。.取得以下结果：（1）单纯股动脉结扎侧和用动-静脉吻合侧的侧支血管之间存在miRNA差异性表达，差异2倍以上的miRNA有94种；其中，上调的miRNA有44种，下调的有50种；（2）原位杂交结果显示miR-352表达于侧支血管内皮细胞和平滑肌细胞，其在高血流切应力情况下表达下调明显；（3）内皮细胞过表达miR-352导致细胞增殖下降、迁移和成管形功能减弱；抑制miR-352使得内皮细胞增殖能力上调、迁移和成管形功能增强；（4）体内抑制miR-352后，侧支血管管径增大、数目增多、管壁各层细胞增殖活跃；荧光微球灌注血流测定血流增加；（5）荧光素酶报告实验证实IGF2R为miR-352的靶基因；（6）培养的内皮细胞过表达miR-352下调IGF2R的表达，反之，抑制miR-352则上调IGF2R的表达；（7）抑制miR-352且同时抑制IGF2R导致内皮细胞的自噬、增殖和成管能力变弱；（8）体内实验，提高血流切应力或敲除miR-352导致侧支血管IGF2R和IGF2表达增加，自噬标志物和溶酶体相关指标表达上调，而Caspase3活性下降；（9）miR-497-5p在吻合侧侧支血管的表达下降；（10）miR-497-5p具有抑制内皮细胞增殖、迁移和成管形等功能；（11）IGF2R是miR-497-5p的靶基因。.结论：（1）动-静脉吻合提高大鼠后肢的血流切应力改变了侧支血管miRNA的表达谱；.（2）miR-352对大鼠主动脉内皮细胞和侧支血管具有抗毛细血管生成及抗动脉生成的作用，miR-352可能是一种新的潜在治疗缺血性疾病的靶向miRNA。.（3）miR-352负性调节内皮细胞和侧支血管IGF2R的表达和自噬；（2）细胞培养实验揭示IGF2R正性调节内皮细胞的自噬。.（4）miR-497-5p通过靶基因IGF2R负性调节内皮管