Investigation of the Functional Roles of MicroRNA-320 in Skeletal Muscle

PROJECT SUMMARY/ABSTRACT Disuse, denervation, and several diseases result in skeletal muscle atrophy and weakness – a major contributor to disease-related disability and death. The mechanisms underlying these processes are incompletely understood, and therapeutics are only emerging. MicroRNAs are known to regulate many important cellular processes. We discovered

that in muscle developing weakness due to disuse (human) and denervation (mouse), there is a marked reduction in microRNA320. Further, bioinformatic analysis demonstrates that FoxOs – a group of transcription factors important in muscle degradation -- are potential targets of miR320. Indeed, when we disrupted miR320 in cultured muscle cells, the FoxO pathway and

protein ubiquitination were upregulated. There is also evidence from others that miR320 may have roles in skeletal muscle metabolism. Given these findings, we created a muscle-specific knockout mouse (miR320mKO) to explore the role of this miRNA in controlling skeletal muscle growth and maintenance in a long-

term, in vivo model. We have found that these mice develop severe muscle wasting over their first 4 months of life. This project will fully characterize the miR320mKO mice at the phenotypic level, investigate the mechanisms by which this phenotype occurs, and carry out a pilot effort at miR320 treatment in a model of mouse muscle atrophy. It promises to open up new avenues in

treatment of skeletal muscle disease. Specifically, we will: 1. Characterize the phenotype of miR320mKO mice -examine muscle size and microscopy, fiber size and type, muscle contractile force in KO vs. controls; examine growth of proliferation and myotube formation of miR320mKO primary muscle cell cultures

2. Investigate the mechanisms underlying the phenotype of miR320mKO mice -dissect operant molecular signaling pathways and events using RNA sequencing; identify potential targets of miR320 by miR-eCLIP and RNA sequencing, and confirm those targets with 3’UTR luciferase reporters in C2C12 cells 3. Carry out a pilot treatment of skeletal muscle wasting with miR320 in mice

-study denervated limb muscle following delivery of miR320 via electroporation vs. controls