Efficient Regulation of VEGF Expression by Promoter-Targeted Lentiviral shRNAs Based on Epigenetic Mechanism A Novel Example of Epigenetherapy
Efficient Regulation of VEGF Expression by Promoter-Targeted Lentiviral shRNAs Based on Epigenetic Mechanism A Novel Example of Epigenetherapy
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DOI：
10.1161/circresaha.109.200774
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发表时间：
2009-09-11
影响因子：
20.1
通讯作者：
Yla-Herttuala, Seppo
中科院分区：
文献类型：
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作者：
Turunen, Mikko P.;Lehtola, Tiia;Yla-Herttuala, Seppo
理由：我们研究了 shRNA 可以通过表观遗传机制在启动子水平导致转录基因激活的可能性。目的：本研究的目的是在体外和体内实验动物中使用稳定的局部慢病毒基因转移来测试启动子靶向小发夹 RNA (shRNA) 对血管内皮生长因子 (VEGF-A) 表达的影响。方法和结果：鉴定了一种 shRNA，它在 C166 内皮细胞中显着增加 VEGF-A 表达。 mRNA 和蛋白质水平，而另一种 shRNA 降低 VEGF-A 表达。定量染色质免疫沉淀分析显示，抑制性 shRNA 引起表观遗传变化，从而增加启动子和转录起始位点内的核小体密度，并导致 VEGF-A 表达的抑制。激活shRNA引起的表观遗传变化与抑制shRNA引起的表观遗传变化相反。这些结果在局部基因转移后的缺血小鼠后肢模型中得到了体内证实，其中通过启动子靶向的 shRNA 实现 VEGF-A 上调，增加了血管分布和血流量。结论：我们表明，慢病毒介导的 shRNA 分子靶向 mVEGF-A 启动子中的特定区域，通过表观遗传调节诱导或抑制 VEGF-A 表达。因此，我们描述了一种基于启动子水平的表观遗传机制的基因治疗新方法，即表观基因治疗。通过操纵特定的表观遗传标记来控制转录为治疗多种疾病提供了一种新方法。 （循环决议 2009；105：604-609。）
Rationale: We studied a possibility that shRNAs can lead to transcriptional gene activation at the promoter level via epigenetic mechanism.Objective: The purpose of this study was to test the effects on vascular endothelial growth factor (VEGF-A) expression by promoter targeted small hairpin RNAs ( shRNAs) in vitro and in experimental animals in vivo using stable local lentiviral gene transfer.Methods and Results: One shRNA was identified which strongly increased VEGF-A expression in C166 endothelial cells at mRNA and protein level whereas another shRNA decreased VEGF-A expression. Quantitative chromatin immunoprecipitation analysis revealed that the repressing shRNA caused epigenetic changes, which increased nucleosome density within the promoter and transcription start site and led to repression of VEGF-A expression. Epigenetic changes caused by the activating shRNA were opposite to those caused by the repressing shRNA. These results were confirmed in vivo in an ischemic mouse hindlimb model after local gene transfer where VEGF-A upregulation achieved by promoter-targeted shRNA increased vascularity and blood flow.Conclusions: We show that lentivirus-mediated delivery of shRNA molecules targeted to specific regions in the mVEGF-A promoter either induce or repress VEGF-A expression via epigenetic modulation. Thus, we describe a new approach of gene therapy, epigenetherapy, based on an epigenetic mechanism at the promoter level. Controlling transcription through manipulation of specific epigenetic marks provides a novel approach for the treatment of several diseases. ( Circ Res. 2009; 105: 604-609.)