摘要
目的筛查动脉粥样硬化闭塞症（ASO）血管重建术后再狭窄相关miRNA基因表达谱，分析并鉴定表达差异显著的miRNA及其潜在靶基因。方法取下肢ASO患者股动脉标本6例，3例未行血管重建术（A组），3例动脉重建术后再狭窄（R组），另取正常股动脉3例为正常对照（N组）。利用miRNA基因芯片检测miRNA差异表达谱，以实时逆转录-聚合酶链反应（real-timeRT-PCR）验证其表达，并通过构建双荧光素酶报告基因系统鉴定预测靶基因。结果与N组比较，病变组（A组＋R组）miRNA-1和miRNA-335表达下调，没有miRNA表达上调；R组miRNA-623表达上调，17个miRNAs下调；A组miRNA-1和miRNA-335表达下调，没有miRNA表达上调。与A组比较，R组17个miRNAs上调，9个miRNAs下调。表达下调以miRNA-1最显著，并经real．timelit．PCR证实（P〈0．05）。双荧光素酶实验证实miRNA-1靶向抑制Kruppel样因子4（KLF4）的表达（P〈0．05）。结论miRNA可能参与ASO及动脉重建术后再狭窄的发病机制，其中miRNA-1可能起重要作用。
Objective To identify artery-specific mircoRNAs （miRNAs） in restenosis after arterial reconstruction in patients with atherosclerosis obliterans （ASO） and validate the target gene of aberrantly expressed miRNA. Methods Superficial femoral artery tissue samples were collected from six patients with ASO and three normal controls who underwent above-knee amputation for limb injury （Group N）. The six patients were divided into two groups based on prior arterial reconstruction： Group A, ASO without re- vascularization （n = 3） and Group R, restenosis following revascularization （n = 3 ）. MiRNA expression profiling of all samples was performed using miRNA microarray analysis, the selected aberrantly expressed miRNAs were validated using real-time reverse transcription-polymerase chain reaction （real-time RT- PCR） analysis and the predicted target gene was assessed using dual-luciferase reporting assays. Results MiRNA microarray analysis showed that miRNA-1 and miRNA-335 were down-regulated whereas there was no miRNA up-regulated in arteries from patient group （Group A and Group R） as compared to those from Group N. In subgroup analyses, miRNA-1 and miRNA-335 were down-regulated whereas there was no miRNA up-regulated as compared to those from Group N; there were 17 miRNAs down-regulated while only hsa-miR-623 up-regulated in arteries from Group R as compared to those from Group N; there were 17 miRNAs up-regulated and 9 miRNAs down-regulated in arteries from Group R as compared to those from Group A. Among all down-regulated miRNAs, down-regulation of miRNA-1 was the largest, and it was confirmed by real-time RT-PCR （ P 〈 0. 05 ）. At the same time, dual-luciferase reporting assay data dem- onstrated that miR-1 targets a putative binding site in the 3 ＇ -UTR of kruppel like factor 4 （ KLF4, P 〈 0. 05）. Conclusion MiRNAs may play important roles in human restenosis following arterial reconstruction, and they may have therapeutic applications in the prevention of neointimal hyperplasia. MiRNA-1 might play a pivotal role in the pathogenesis of both atherosclerotic ASO and restenosis.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第3期535-538,共4页
Chinese Journal of Experimental Surgery
基金
上海市浦江人才计划基金资助项目
