Genetic deletion of microRNA biogenesis in muscle cells reveals a hierarchical non- clustered network that controls focal adhesion signaling during muscle regeneration
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. Overall design: We performed genome-wide identification of differentially expressed genes and miRNA target regulation after single or combinatorial miRNA inhibition in human myotubes. Human myoblasts derived from 3 donors were treated with control antagomirs or antagomirs either against let-7, miR-29a, miR-125b, miR-199, miR-221 or against all miRNAs (ant-5x). Twenty-four hours after transfection differentiation was induced for two days and total RNA was isolated. Libraries were prepared using TruSeq Stranded mRNA Library Prep kit (Illumina) and samples were sequenced using NovaSeq 100bp single-end sequencing at the Functional Genomics Center Zurich.