A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.
A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.
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DOI：
10.1038/gt.2016.22
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发表时间：
2016-06
期刊：
影响因子：
5.1
通讯作者：
Amendt BA
中科院分区：
文献类型：
--
作者：
Cao H;Yu W;Li X;Wang J;Gao S;Holton NE;Eliason S;Sharp T;Amendt BA
目前用于抑制microRNA（miR）功能的工具仅限于修饰的反义寡核苷酸、海绵和诱饵RNA分子，并且没有一种用于理解发育期间的miR功能。由于重复，CRISPR/Cas介导的基因组内miR序列的缺失需要多个染色体缺失以去除所有功能性miR家族成员。在这里，我们报告了一种新的基于质粒的miR抑制剂系统（PMIS），该系统表达一种新的RNA分子，可抑制细胞和小鼠中的miR家族成员。PMIS工程化的RNA最佳二级结构、侧翼序列和特异性反义miR寡核苷酸序列以稳定的复合物结合miR以抑制miR活性。在细胞中，一种PMIS可以有效抑制共享相同种子序列的miR家族成员。PMIS显示无脱靶效应或毒性，并且对共享相同种子序列的miR具有高度特异性。表达PMIS-miR-17-18和PMIS-miR-19-92的转基因小鼠显示出与miR-17-92敲除小鼠相似的表型。有趣的是，仅表达PMIS-miR-17-18的小鼠具有与仅表达PMIS-miR-19-92的小鼠不同的发育缺陷，证明PMIS系统在剖析簇内miR的不同功能方面的有用性。不同的PMIS miR抑制剂可以连接在一起以敲低从不同染色体表达的多个miR。对miR-17-92、miR-106 a-363和miR-106 b-25簇的抑制揭示了这些miR的新机制和发育缺陷。我们报告了一种新的工具，可以在没有基因组编辑的情况下剖析miR在发育中的作用，抑制细胞中的miR功能，并作为一种潜在的新治疗试剂。
Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas-mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR family members because of duplications. Here, we report a novel plasmid-based miR inhibitor system (PMIS) that expresses a new RNA molecule, which inhibits miR family members in cells and mice. The PMIS engineered RNA optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. The PMIS shows no off-target effects or toxicity and is highly specific for miRs sharing identical seed sequences. Transgenic mice expressing both PMIS-miR-17-18 and PMIS-miR-19-92 show similar phenotypes of miR-17-92-knockout mice. Interestingly, mice only expressing PMIS-miR-17-18 have developmental defects distinct from mice only expressing PMIS-miR-19-92 demonstrating usefulness of the PMIS system to dissect different functions of miRs within clusters. Different PMIS miR inhibitors can be linked together to knock down multiple miRs expressed from different chromosomes. Inhibition of the miR-17-92, miR-106a-363 and miR-106b-25 clusters reveals new mechanisms and developmental defects for these miRs. We report a new tool to dissect the role of miRs in development without genome editing, inhibit miR function in cells and as a potential new therapeutic reagent.