P.498 - Dystrophin Dp427 is lost due to multiple DMD intron retentions in rhabdomyosarcoma CRL-2061 cells

Abstract

Dystrophin inactivation, responsible for Duchenne muscular dystrophy (DMD), is recently implicated as an anti-tumor suppressor factor in cancers with myogenic characteristics. Rhabdomyosarcoma (RMS), a mesodermal cancer, shows myogenic features and occurs at a higher frequency in mdx mouse, a DMD model mouse. In humans, as the clinical management of DMD advances to increase the life expectancy of patients, a subsequent issue is anticipated to be that of addressing other complications such as RMS. RMS, although displays myogenic properties, has not yet been fully evaluated for dystrophin expression. Here, we used reverse transcription PCR and western blotting to analyze dystrophin in RMS CRL-2061 cells. The 14 kb DMD transcript was fully amplified as 20 fragments as in skeletal muscle. Alternatively, spliced transcripts including 3 intron retentions and 2 exons skipping events were disclosed. In one fragment extending from exon 70 to 79, no normally spliced product was obtained. Instead, 6 alternatively spliced products including a new product deleting exon 73 were identified, and exon 78 deleted products was most abundant. However, dystrophin Dp427 was not detected by western blotting. Remarkably, analysis of 9 short DMD introns indicated retention of introns 40, 58 and 70. These intron retentions occupied 75, 15 and 20 % of the total PCR amplified products, respectively, indicating the possibility of every DMD transcript to carry an intron retention event. Multiple intron retentions in the DMD transcript are suggested to hamper translation of the Dp427 isoform and promote tumorigenesis in RMS. This study exemplifies the important significance of DMD intron retention in tumorigenesis in RMS.