SKELETAL MUSCLE AND CIRCULATING MICRORNAS IN MYOTONIC DISTROPHY TYPE 1

In Myotonic Dystrophy type 1 (DM1), the accumulation of transcripts containing CUG triplet expansions leads to the dysregulation of multiple cellular processes, including RNA splicing and microRNA (miRNAs) synthesis. This project aims to find functional miRNAs that are specifically deregulated in muscles of DM1 patients, and to identify circulating miRNAs in the plasma of DM1 patients that could be used as biomarkers of DM1 disease progression. Preliminary studies had previously identified a group of miRNAs that were deregulated in the plasma or serum of small groups of DM1 patients. In this work, very stringent selection and normalization criteria were adopted to validate or disprove these miRNAs in 103 DM1 patients and 111 matched controls. We found that 8 miRNAs out of 12 were significantly deregulated in DM1 patients and that the levels of these miRNAs discriminated DM1 from controls significantly. Next, we determined a “DM1-miRNAs score”, calculated averaging the values of all 8 miRNAs, and a “miR-133a/b score” obtained averaging the levels of the two miR-133 members, both displaying a good ability to discriminate between DM1 and control subjects. Additionally, both scores correlated with muscle strength and Creatine Kinase levels. Moreover, 7 out of 8 miRNAs were found deregulated also in 30 patient affected by Myotonic Dystrophy type 2. The second aim of this work was the identification of miRNA/target mRNAs couples deregulated in skeletal muscles of DM1 patients. To this aim, the RNAs associated to RISC effector complex were analyzed from muscle biopsies of DM1 patients and healthy individuals and RNA-Sequencing was used to identify RISC-associated small RNAs (miRNAs) and long RNAs (mRNAs). A number of small and long RNAs differentially expressed in RISC complexes of DM1 vs healthy controls were identified, that showed statistically significant modulation. Next, using target prediction algorithms, miRNA-mRNA couples deregulated in DM1 were identified. RNA-sequencing data confirmed in independent qPCR assays, analyzing larger groups of DM1 patients and controls. Moreover, using myogenic cell models derived from dermal fibroblasts of DM1 patients and healthy individuals, a subset of these interactions was confirmed, validating this in vitro system for future functional studies. In conclusion, this study identified relevant tissue miRNAs aberrantly expressed in DM1 and validated plasma miRNAs as potential DM1 biomarkers.