The Role of MyomiRs Oxidation on Muscle Wasting in Ageing and Cancer Cachexia

Abstract

Progressive loss of muscle mass and strength is a hallmark of ageing (sarcopenia) and disease, such as cancer (cachexia). Muscle atrophy and weakness are associated with increased risk of falls, frailty, worse cancer patient outcomes, and increased morbidity and mortality. The mechanisms of muscle loss are not fully understood due to complexity. Various cellular mechanisms have been demonstrated to underlie muscle loss, including changes in the expression of multiple genes. MicroRNAs (miRs), post-transcriptional gene expression regulators, are emerging as important regulators of skeletal muscle homeostasis, particularly due to their simultaneous targeting of multiple gene targets, supporting their role in regulating complex disorders like muscle loss. One of the hallmarks of ageing and cachexia is oxidative stress. Whilst DNA, protein, and lipid oxidation have been characterised in muscle ageing and disease, much less is understood about RNA oxidation, and in particular microRNA oxidation. RNA is more sensitive to oxidative damage than DNA, and products of RNA oxidation (8-oxo-guanine) have been detected in different conditions associated with muscle loss. microRNA oxidation could affect how microRNAs regulate their targets, as 8-oxo-guanine can bind to adenosine as well as uracil—this could dysregulate microRNA ability to bind to its targets or even acquire new targets. To date, oxidation of miRs has been demonstrated during cardiac disease. Oxidised miR-184 was implicated in promoting apoptosis in cardiomyocytes through targeting genes that the native miR-184 does not target, while oxidised miR-1 was associated with the development of cardiomyopathy through target misregulation. However, the oxidation of microRNAs, or the functional consequences of this process, has not been investigated in muscle before. This research examined whether microRNAs are oxidised in muscle during ageing and cancer cachexia and whether oxidised miRs contribute to muscle loss. I hypothesised that oxidation of miRs contributes to muscle loss during ageing and cachexia through dysregulation of microRNA gene targeting. This hypothesis was first tested in muscle samples from older people and mouse models of ageing and lung cancer cachexia. In muscle from older people and old and cachectic mice, differential expression and oxidation of miRs were demonstrated, and the functional consequences were further validated using C2C12 mouse muscle cells and immortalised human muscle cells. Small RNA sequencing identified several oxidised miRs, including miR-1, miR-133, and miR-206. These oxidised myomiRs, muscle-enriched miRs, had pronounced effects on myotube size: exposure of C2C12 myotubes to oxidised myomiRs resulted in changes in myotube size and altered the expression of multiple genes, including those associated with oxidative stress, protein degradation, and autophagy. In addition to human myotubes, myotube size and the protein levels associated with oxidised RNA were decreased. This supports the hypothesis that oxidised miRs, particularly myomiRs: miR-1, miR-133, and miR-206, may partially contribute to muscle atrophy through their regulation of key cellular processes: autophagy and oxidative stress response.