Investigating the subcellular localisation and function(s) of dystrophin protein Dp71 isoforms in glioma

The Dp71 protein is the most expressed product of the DMD gene in the nervous system. Mutation in the region codes dystrophin protein (Dp71) linked to cognitive disturbances in Duchenne muscular dystrophy (DMD) patients. There is growing evidence that the gene is contributing to the development of Central Nervous System related cancers. The aim of this study is to characterise the role of the main 4 Dp71 isoforms by investigating its subcellular localisation and putative cellular functions in glioblastoma cells, the most aggressive and common type of glioma. By transfecting the four GFP-Dp71 constructs into a well characterised human glioblastoma cell line – U251-MG. Immunoblotting was used to assess Dp71 expression in human glioblastoma cell line. Moreover, we examined the subcellular localisation and the effect of Dp71 over expression on the nuclear Lamin B1, cell migration immunofluorescence, and scratch assay. A 71 kDa endogenous Dp71 was expressed in all glioblastoma cells and only GFP-Dp71a (99 kDa) isoform was overexpressed in the transfected cells. Lower Lamin B1 fluorescence intensity and abnormal nuclear shape was observed in cells overexpressing GFP-Dp71a. Furthermore, cytoplasmic and nuclear localisation of Dp71 isoform was found in both cytoplasm and nucleus, but higher in the nucleus. Overexpression of Dp71a transfected cells reduced the cell migration and covering the scratched tissue gaps with 5% and 6%, whilst that of the control cells were 29% and 50% at time 24 and 48 hours, respectively compared to the that at time 0. Dp71ab transfected cells showed similar cell migration to the control. We concluded that Dp71 overexpression had a clear effect on the expression of Lamin B1 and cell migration. Additionally, localisation of Dp71a was higher in the nucleus than in the cytoplasm