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Genetic deletion of microRNA biogenesis in muscle cells reveals a hierarchical non-clustered network that controls focal adhesion signaling during muscle regeneration.
Molecular Metabolism ( IF 8.1 ) Pub Date : 2020-03-05 , DOI: 10.1016/j.molmet.2020.02.010
Edlira Luca 1 , Katarina Turcekova 2 , Angelika Hartung 1 , Sebastian Mathes 3 , Hubert Rehrauer 4 , Jan Krützfeldt 5
Affiliation  

Objective

Decreased muscle mass is a major contributor to age-related morbidity, and strategies to improve muscle regeneration during ageing are urgently needed. Our aim was to identify the subset of relevant microRNAs (miRNAs) that partake in critical aspects of muscle cell differentiation, irrespective of computational predictions, genomic clustering or differential expression of the miRNAs.

Methods

miRNA biogenesis was deleted in primary myoblasts using a tamoxifen-inducible CreLox system and combined with an add-back miRNA library screen. RNA-seq experiments, cellular signalling events, and glycogen synthesis, along with miRNA inhibitors, were performed in human primary myoblasts. Muscle regeneration in young and aged mice was assessed using the cardiotoxin (CTX) model.

Results

We identified a hierarchical non-clustered miRNA network consisting of highly (miR-29a), moderately (let-7) and mildly active (miR-125b, miR-199a, miR-221) miRNAs that cooperate by directly targeting members of the focal adhesion complex. Through RNA-seq experiments comparing single versus combinatorial inhibition of the miRNAs, we uncovered a fundamental feature of this network, that miRNA activity inversely correlates to miRNA cooperativity. During myoblast differentiation, combinatorial inhibition of the five miRNAs increased activation of focal adhesion kinase (FAK), AKT, and p38 mitogen-activated protein kinase (MAPK), and improved myotube formation and insulin-dependent glycogen synthesis. Moreover, antagonizing the miRNA network in vivo following CTX-induced muscle regeneration enhanced muscle mass and myofiber formation in young and aged mice.

Conclusion

Our results provide novel insights into the dynamics of miRNA cooperativity and identify a miRNA network as therapeutic target for impaired focal adhesion signalling and muscle regeneration during ageing.



中文翻译:

肌肉细胞中microRNA生物发生的遗传缺失揭示了一个分层的非簇状网络,该网络控制着肌肉再生过程中的粘着信号传导。

目的

肌肉质量下降是与年龄相关的发病率的主要因素,因此迫切需要在衰老过程中改善肌肉再生的策略。我们的目标是鉴定参与肌肉细胞分化关键方面的相关microRNA(miRNA)的子集,而与miRNA的计算预测,基因组聚类或差异表达无关。

方法

使用tamoxifen可诱导的CreLox系统删除了原代成肌细胞中的miRNA生物发生,并结合了回送的miRNA文库筛选。在人类原代成肌细胞中进行了RNA-seq实验,细胞信号转导事件和糖原合成以及miRNA抑制剂。使用心毒素(CTX)模型评估了年轻和老年小鼠的肌肉再生。

结果

我们确定了一个分层的非集群miRNA网络,该网络由高度(miR-29a),中等(let-7)和轻度活跃(miR-125b,miR-199a,miR-221)的miRNA组成,这些miRNA通过直接靶向焦点粘附复合物。通过比较miRNA单一抑制和组合抑制的RNA-seq实验,我们发现了该网络的基本特征,即miRNA活性与miRNA协同作用成反比。在成肌细胞分化过程中,五个miRNA的组合抑制作用增加了粘着斑激酶(FAK),AKT和p38丝裂原活化蛋白激酶(MAPK)的活化,并改善了肌管形成和胰岛素依赖性糖原合成。此外,

结论

我们的结果为miRNA协同动力学提供了新颖的见解,并确定了miRNA网络是衰老期间粘着斑信号传导和肌肉再生受损的治疗目标。

更新日期:2020-03-05
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