MiR-199a-5p抑制下肢动脉硬化闭塞支架成形术后再狭窄的作用和机制研究

Excessive artery smooth muscle cell (ASMCs) proliferation and migration play pivotal steps in the process of In-Stent Restenosis (ISR) of arteriosclerosis obliterans (ASO) after stent implantation. The incidence of ISR is 30%-50% of ASO population after stent implantation in 1 year. However, the underlying mechanism remains unclear. Drug Eluting Balloon (DEB) or Drug Eluting Stents (DES) could reduce the early-term ISR rate by inhibiting ASMCs proliferation and migration with coated drugs (generally anti-proliferative agents like Paclitaxel or Sirolimus). However, the coated drug is less cell-specific that also induces endothelial cell (EC) apoptosis and impairs the process of re-endothelialization, leading to late thrombosis or atherosclerotic occlusion.. MicroRNA is recognized as an important modulator of ASMCs phenotype by regulating target gene expression at post-transcriptional level. Previously, we reported the microRNA expression profile of ASO samples and identified that hypoxia inducible factor 1α (HIF-1α)/miR-21/Tropomyosin1 axis played an essential role in ASMCs phenotype modulation that lead to ASO genesis and progression. However, whether microRNA is involved into the process of ISR after stenting angioplasty in ASO remains unknown. We first profiled the microRNA expression signature of ISR samples after stent implantation. Among the dysregulated microRNAs, miR-199a-5p was found most significantly downregulated in the smooth muscle layer of ISR samples. Further introducing miR-199a-5p into primary cultured ASMCs using miR-199a-5p mimic transfection significantly attenuated mitogens stimulated ASMCs proliferation. It indicates that loss of miR-199a-5p might be related with ISR after stent implantation. However, the exact role and underlying mechanisms of miR-199a-5p regulating ASMCs phenotype switch in ISR remains unknown. Interestingly, miR-199a-5p inhibited ASMCs proliferation, but induce EC apoptosis of impairs EC migration. We speculated that miR-199a-5p inhibits ASMCs proliferation and migration via targeting HIF-1α/miR-21 and E2F3, on the other hand, miR-199a-5p preserves EC function via targeting p27Kip1, and induces ASMCs to recruit EC via targeting DANJA4/ApoE, thus in total, miR-199a-5p attenuates the ISR process. It suggest that miR-199a-5p might be an ideal potential targets for preventing ISR.. The expression primary miR-199a-1/2 is downregulated in ISR, indicating that miR-199a-5p is regulated by DNA methylation. In ISR samples, the expression DNA methyltransferase (DNMT) significantly increased. Intriguingly, treating ASMCs with demethylation drug (5-AZA) significantly increased miR-199a-5p expression. Thus we hypothesize that promoter DNA hypermethylation results miR-199a-5p downregulation.. In the present study, we intend to use miR-199a-5p mimic and inhibitor transfection, Edu incorporation and transwell assay to determine the role of miR-199a-5p in regulating ASMCs phenotype switch. Evaluate the therapeutic role of miR-199a-5p in treating ISR by using lentivirus mediated overexpression in animal models. Use dual-luciferase assay to achieve the critical evidence of miR-199a-5p directly binds HIF-1α, E2F3 and DNAJA4 mRNA 3'-UTR. Determine the HIF-1α/miR-21 involvement into miR-199a-5p conducted ASMCs phenotype switch, and the correlation between miR-199a-5p and HIF-1α in ASO samples. Further using BSP sequencing and MethyLight analysis to reveal the methylation status of miR-199a-5p promoter in ISR. . The present study would shed new light on the possible role and mechanism of miR-199a-5p modulating ASMCs and EC in the process of ISR, and may provide more sufficient evidence for preventing ISR.

动脉平滑肌细胞（ASMCs）过度增殖、迁移是导致下肢动脉硬化闭塞（ASO）支架成形术后再狭窄（ISR）的主要原因，其机制尚不明确。药物洗脱球囊/支架缺乏特异性干预靶点，过多损伤ASMCs和内皮细胞（EC），导致内皮修复受抑制，影响远期效果。我们预实验发现，ISR的平滑肌层中miR-199a-5p表达显著下调，上调其表达后可明显抑制ASMCs增殖，且不导致EC损伤，其中的机制有待于阐明。据此我们提出科学假说：上调miR-199a-5p一方面可通过靶向抑制HIF-1α/miR-21通路以及转录因子E2F3的表达，抑制ASMCs增殖、迁移；另一方面可通过靶向沉默p27Kip1保护EC功能，以及通过抑制DNAJA4/ApoE来诱导ASMCs募集EC，加速内皮化，两方面协同作用抑制ISR进程。本项目将阐明miR-199a-5p通过抑制ASMCs和保护EC，防治ISR的新机制，为干预ISR提供新靶点。

动脉平滑肌细胞（ASMCs）过度增殖、迁移是导致下肢动脉硬化闭塞（ASO）术后再狭窄（RS）的主要原因，现有腔内载药器具缺乏特异性，过多诱发细胞凋亡内皮修复受损，导致患者死亡率升高。本课题发现，在正常动脉中miR-199a-5p高表达，而在RS中表达显著减少。上调miR-199a-5p表达后可明显抑制ASMCs增殖和迁移，减少S期的细胞数量，促进细胞凋亡；反之则促进ASMCs增殖迁移，增加S期的细胞数量，减少细胞凋亡。生物信息学分析发现，E2F3和HIF-1α mRNA的3’UTR区域含有miR-199a-5p的种子序列，升高miR-199a-5p可显著降低ASMC中E2F3和HIF-1α的蛋白表达；而抑制miR-199a-5p可明显促进E2F3和HIF-1α的蛋白表达；荧光素酶基因报告法证实，miR-199a-5p可直接结合E2F3和HIF-1α mRNA 3’UTR区域。采用慢病毒载体进行回复实验，证实E2F3和HIF-1α直接参与了miR-199a-5p对ASMCs的调控。在动物模型中，升高miR-199a-5p水平可明显抑制动脉球囊扩张成形后RS，但没有延缓内皮化。在内皮细胞（EC）中，升高miR-199a-5p水平对细胞增殖、迁移和成管能力没有明显影响，而降低miR-199a-5p水平可明显损害EC的成管能力。升高miR-199a-5p可明显抑制EC中DNAJA4和p27Kip1的蛋白表达，反之则促进DNAJA4和p27Kip1的蛋白表达，荧光素酶基因报告法证实miR-199a-5p可与DNAJA4和p27Kip1 mRNA的3’UTR区域结合。采用慢病毒载体进行回复实验，证实miR-199a-5p对EC的保护作用是通过靶向沉默DNAJA4和p27Kip1 mRNA完成。在动物模型和临床样本中证实，E2F3、HIF-1α、DNAJA4和p27Kip1的表达水平与miR-199a-5p成明显负相关。最终确定了miR-199a-5p的启动子范围，证实其受DNA甲基化的调控。本项目阐释了miR-199a-5p通过靶向E2F3和HIF-1α抑制ASMCs、减轻动脉球囊扩张成形后再狭窄的新机制，并进一步深入探讨了miR-199a-5p通过靶向DNAJA4和p27Kip1保护EC的新机制，为干预ASO术后ISR提供了潜在的新靶点。