Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany.
RNA Biol. 2012 Aug;9(8):1088-98. doi: 10.4161/rna.21165. Epub 2012 Aug 1.
Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17-5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3'-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications.
反义抑制致癌或其他疾病相关的 miRNA 和 miRNA 家族在体内可能提供新的治疗策略。然而，这种方法依赖于强有力的 miRNA 抑制剂的开发及其有效递送到细胞中。在这里，我们引入具有磷酸二酯骨架（PO）的短种子导向的 LNA 寡核苷酸（12 或 14 个碱基的反义种子），用于有效的 miRNA 抑制。我们已经使用 let-7a 控制的荧光素酶报告基因测定分析了这种 LNA（PO）反义种子，并在体外将其鉴定为有效的 miRNA 抑制剂。此外，针对致癌的 miR-17-5p 和 miR-20a 的 LNA（PO）14 个碱基反义种子比对应的 miRNA 发夹抑制剂更持久地解除内源性 p21 的表达抑制，后者通常用于抑制 miRNA 功能。进一步分析反义种子介导的 p21 在荧光素酶报告构建体中的去抑制作用-包含 p21 的 3'UTR 并含有两个 miRNA 结合位点，miR-106b 家族-提供了证据表明 LNA 反义种子抑制 miRNA 家族，而发夹抑制剂以 miRNA 特异性方式作用。通过对 miR-106b 靶位点的种子诱变，证明了 LNA 反义种子引起的去抑制作用是特异性的。重要的是，我们展示了 LNA（PO）14 个碱基反义种子与聚乙烯亚胺（PEI F25-LMW）复合后递送到细胞中的功能，这导致了聚合物纳米颗粒的形成。相比之下，试图通过与聚乙烯亚胺（PEI F25-LMW）制剂来递送电中性的靶向微小的 LNA 8 个碱基的带有硫代磷酸酯骨架（PS）的功能性种子导向 tiny LNA 8 个碱基的尝试仍然没有成功。总之，LNA（PO）14 个碱基反义种子是有吸引力的 miRNA 抑制剂，其基于 PEI 的递药可能代表治疗应用的有前途的新策略。
