Circular RNA role in Myotonic Dystrophy type 1

Introduction Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the 3? UTRof the DMPK gene leading to deregulated mRNA-splicing. Circular RNAs (circRNAs), a class of covalentlyclosed RNAs generated by back-splicing events, are emerging as important regulators of muscular disorders.Given the pronounced splicing dysregulation in DM1, we hypothesized that circRNA levels and function maybe deregulated in DM1. Accordingly, we have previously identified a small subset of circRNAs that weresignificantly increased in muscle biopsies of DM1 patients. Results By analysing available RNA-seq datasets of DM1 patients and applying a stringent selection pipeline, weidentified 20 additional circRNAs candidates differentially expressed between DM1 and CTRLs and displayinga high ratio between the circular and the linear isoforms. Validation of DM1-circRNAs was performed inbiceps brachii biopsies of 24 DM1 and 16 CTRL matched subjects. RT-qPCR analysis using divergent andconvergent primers allowed to assess the levels of the circular and linear isoforms of candidate genes. Thecircular structure of DM1-circRNAs was confirmed by Sanger sequencing and resistance to the RNase Rdigestion. This way, we identified 6 new DM1-circRNAs displaying ROC curves that efficiently discriminatedDM1 patients from controls, suggesting a potential role as disease biomarkers. In particular, circARHGAP10displayed a significant direct correlation with the number of CTG-repeats, and an inverse correlation withskeletal muscle strength of DM1 patients. Bioinformatics prediction of miRNA/circRNAs binding indicatesthat circARHGAP10 may act as a sponge for miR-409-3p, a miRNA associated with mitochondrial damage indifferent muscular dystrophies. Indeed, experiments using human DM1 myogenic cell lines showed that miR-409-3p was enriched in a pull-down assay using biotinylated oligonucleotides targeting circARHGAP10backsplice junction. In addition, Ago-2 RISC IP analysis confirmed that circARHGAP10 could serve as abinding platform for miR-409-3p. Accordingly, in vitro, circARHGAP10 specific silencing inhibited miR-409-3p expression, while, in DM1 patients, miR-409-3p levels were increased in muscle tissue. Conclusion New circRNAs dysregulated in DM1 patients that might be used as DM1 biomarkers were identified. Inaddition, we found that hsa-miR-409-3p might be a potential target for circARHGAP10.