Reducing muscleblind protein levels causes decreased DMPK transcripts and a muscle-specific chloride channel in myotonic dystrophy type one cell line

Abstract

From a molecular perspective, myotonic dystrophy (DM) is characterised by the presence of abnormally long units of CUG and CCUG repeats that interact with some specific nuclear proteins to form foci in patient’s cells. It leads to entrapment of repeats in the nuclei to form foci in DM cells due to its association with muscleblind proteins. MBNL proteins also direct the alternative splicing of a group of genes that are aberrantly spliced in DM. In this study, MBNL downregulation was performed using short interfering RNA morpholinos designed against muscleblind (MBNL) proteins and applied to DM1 cells, as well as the wild-type and DM1 cells with a transgene splicing construct from a previous study, to assess the effect of its reduction on splicing, as well as the formation of foci in DM1 cells. The results indicate that MBNL downregulation had no effect on transgene splicing construct in wild-type cells as it showed a fluorescence ratio of 0.289 for both double downregulated MBNL and scrambled knockdown, while the untreated had a ratio of 0.228. In DM1 cells, the fluorescence ratio for the splicing construct was 0.174 for MBNL downregulation, 0.250 for scrambled and 0.251 for untreated cells. Additionally, MBNL downregulation affected foci reduction to 59.3±5.7% compared to 99.3±5.7% for scrambled. Furthermore, MBNL downregulation induced a decrease in mutant DMPK transcript, which was 18.0%, while scrambled and untreated were 38.4 and 53.3%, respectively. Conclusively, results from this study indicate that MBNL proteins, along with mutant DMPK transcript, could serve as a molecular target for therapy in myotonic dystrophy.