Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells.
Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells.
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DOI：
10.1186/s12967-017-1273-x
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发表时间：
2017-08-02
影响因子：
7.4
通讯作者：
Miller JL
中科院分区：
文献类型：
--
作者：
de Vasconcellos JF;Byrnes C;Lee YT;Allwardt JM;Kaushal M;Rabel A;Miller JL
在人类中，由RNA结合蛋白LIN28及其主要靶点let-7家族的microRNAs(MiRNAs)组成的异时级联反应在人类红系个体发育过程中受到高度调控。此外，培养的成人CD34(+)细胞中let-7miRNAs的下调或HbSS基因儿童患者培养的红细胞中LIN28的过度表达会导致胎儿血红蛋白(HBF)水平升高，范围为总水平的19%-40%。因此，我们假设，针对单个let-7miRNA家族成员的靶向对成人红细胞中HBF的表达具有调节作用。用RT-qPCR方法检测外周血分离纯化的细胞群体中成熟的let-7家族成员的表达水平。为了研究let-7miRNAs对珠蛋白表达的影响，将含有针对let-7a或let-7b的TUD设计的慢病毒构建物与空载体对照进行了比较。将成年健康志愿者的CD34(+)细胞在添加促红细胞生成素的无血清培养液中体外培养21天后进行转导。下游分析包括逆转录定量聚合酶链式反应(RT-qPCR)、蛋白质印迹(Western印迹)和高效液相色谱(HPLC)，用于鉴定成人和胎儿的血红蛋白。单个let-7miRNA家族成员在成人外周血细胞中的表达表明，在纯化的成人血细胞亚群中，let-7a和let-7b miRNAs的表达水平明显高于其他let-7家族成员，且主要在网织红细胞中表达。因此，本研究采用TUD设计对let-7a和let-7b的靶向抑制作用进行研究，以探讨其对发育调控红系基因的影响。LET-7a-TUD转导显著增加了γ-珠蛋白mRNA的表达和HbF，平均增加38%。Let-7a-TUD还显著降低了一些个体发育调控的红系基因CA1和GCNT2的mRNA表达。此外，LET-7a-TUD下调和上调了与红系相关的转录因子BCL11A和HMGA2，而ZBTB7A、KLF1和SOX6没有变化。总体而言，我们的数据表明，let-7 miRNAs在人类造血细胞中有差异表达，并且对该家族高表达物种的靶向抑制足以导致伽马珠蛋白表达和HBF水平的发育特异性变化。本文的在线版本(doi：10.1186/s12967-0171273-x)包含补充材料，授权用户可以使用。
In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19–40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels. The online version of this article (doi:10.1186/s12967-017-1273-x) contains supplementary material, which is available to authorized users.
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影响因子：
14.9
作者：
Kozomara A;Griffiths-Jones S
通讯作者：
Griffiths-Jones S
影响因子：
3.7
作者：
Lee YT;de Vasconcellos JF;Byrnes C;Kaushal M;Rabel A;Tumburu L;Allwardt JM;Miller JL
通讯作者：
Miller JL
影响因子：
14.9
作者：
Griffiths-Jones, S
通讯作者：
Griffiths-Jones, S
Mirbase：MicroRNA基因组学的工具。
影响因子：
14.9
作者：
Griffiths-Jones, Sam;Saini, Harpreet Kaur;van Dongen, Stijn;Enright, Anton J.
通讯作者：
Enright, Anton J.
DOI：
10.1126/science.aad3312
发表时间：
2016-01-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Masuda T;Wang X;Maeda M;Canver MC;Sher F;Funnell AP;Fisher C;Suciu M;Martyn GE;Norton LJ;Zhu C;Kurita R;Nakamura Y;Xu J;Higgs DR;Crossley M;Bauer DE;Orkin SH;Kharchenko PV;Maeda T
通讯作者：
Maeda T