Investigating microRNA-target interactions during skeletal muscle development in chicken embryos
Investigating microRNA-target interactions during skeletal muscle development in chicken embryos
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发表时间：
2017-05
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通讯作者：
Camille Viaut
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作者：
Camille Viaut
MicroRNA（miRNAs）是一类短的非编码RNA，在转录后调节基因表达，具有广泛的意义，参与了包括肌肉疾病在内的多种生物学过程。除了myomiRs（其是在横纹肌中高度富集的miRNAs）之外，测序技术和生物信息学的最新进展导致在脊椎动物和其他物种中鉴定出大量miRNAs。然而，对于许多这些miRNAs的具体作用，特别是在肌生成过程中，尚未确定。在这里，我研究了miR-128的潜在功能，证实了与其候选靶点之一Eya 4的相互作用，并研究了其敲低对鸡胚胎骨骼肌发生的影响。通过LNA原位杂交（LNA ISH）表征miR-128以及其他22种体细胞miRNA的表达模式。通过计算分析，Eya 4被鉴定为miR-128的候选“肌肉”靶标。其表达模式的特点; miR-128和Gga-Eya 4在发育体节中显示出相似的特征。使用米兰达算法，在其他转录因子的3'非翻译区（UTR）中鉴定了潜在的miRNA结合位点，这些转录因子沿着Eya 4是PAX-SIX-EYA-DACH（PSED）网络的成员（Six 1/4、Eya 1/2/3和Dach 1）。在体外和体内检查这些miRNA/靶标相互作用。通过荧光素酶报告基因测定证实Gga-Eya 4是miR-128以及miR-206的靶标。在鸡胚胎中注射miR（AM）-128后，通过RNA ISH和RT-qPCR验证miR-128/Gga-Eya 4相互作用。miR-128的敲低导致Gga-Eya 4表达的显著去抑制;还观察到Gga-Six 4和Gga-Pax 3表达的增加，而Gga-MyoD 1表达降低。通过这个项目，使用基于细胞的实验和动物研究的组合，我表明miR-128可以通过靶向PSED网络的成员Gga-Eya 4在鸡胚胎骨骼肌发生的调节中发挥重要作用。
MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance and have been implicated in many biological processes during development and disease, including muscle disease. In addition to the myomiRs, which are miRNAs highly enriched in striated muscles, recent advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species. However, for many of these miRNAs specific roles, in particular during myogenesis, have not yet been determined. Here, I investigated the potential functions of miR-128, confirmed an interaction with one of its candidate targets, Eya4, and looked at the impact of its knock-down on skeletal myogenesis in the chicken embryo. The expression pattern of miR-128, as well as 22 other somitic miRNAs, were characterised by LNA in situ hybridisation (LNA ISH). Eya4 was identified as a candidate ‘muscle’ target of miR-128 by computational analysis. Its expression pattern was characterised; miR-128 and Gga-Eya4 displayed similar profiles in developing somites. Using the miRanda algorithm potential miRNA binding sites were identified in the 3’ untranslated region (UTR) of other transcription factors, which along with Eya4 are members of the PAX-SIX-EYA-DACH (PSED) network (Six1/4, Eya1/2/3, and Dach1). These miRNA/target interactions were examined in vitro and in vivo. Gga-Eya4 was confirmed as a target of miR-128 as well as miR-206 by luciferase reporter assays. MiR-128/Gga-Eya4 interaction was validated by RNA ISH and RT-qPCR after antagomiR (AM)-128 injection in chicken embryos. Knock-down of miR-128 resulted in a significant de-repression of Gga-Eya4 expression; an increase in Gga-Six4 and Gga-Pax3 expression was also observed, whereas Gga-MyoD1 expression was decreased. With this project, using a combination of cell-based experiments and animal studies, I showed that miR-128 could play an important role in the regulation of skeletal myogenesis in the chicken embryo by targeting Gga-Eya4, a member of the PSED network.