2016 Fiscal Year Research-status Report
Dystrophin intron retention analysis to identify new targets for Antisense Oligonucleotide mediated RNA modulation in Rhabdomyosarcoma
| Project/Area Number |
16K07216
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| Research Institution | Kobe Gakuin University |
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Principal Investigator |
ニバ タベ・エマ・エコ 神戸学院大学, 総合リハビリテーション学部, 研究員 (00727810)
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| Co-Investigator(Kenkyū-buntansha) |
松尾 雅文 神戸学院大学, 総合リハビリテーション学部, 教授 (10157266)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | Rhabdomyosarcoma / Dystrophin / Intron retention |
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| Outline of Annual Research Achievements |
Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma in the world arising from skeletal muscle progenitors. Malfunction of the Duchenne muscular dystrophy-related gene, dystrophin was implicated in the growth and metastasis of tumor in RMS.
In this project, the applicants attempted to study dystrophin intron retention in RMS to identify targets for RNA modulation by Antisense Oligonucleotides (AO).
This year, the applicants successfully confirmed the retention of intron 40 and 58 in a variety of RMS cell lines using primers to amplify exons 40 to 41 and 57 to 59 regions respectively. Next, they performed sequencing analysis to confirm the nucleotide arrangement and size of the sequence of intron 40 (851bp) and 58 (608bp), respectively. From the result, intron 40 retention was registered in 2 of 3 RMS cells in two patterns; retention of the whole of intron 40 and/or the 3’ end of intron 40 (exon 41E), respectively. Direct sequencing analysis also showed retention of whole intron 58 in RMS. In addition to introns 40 and 58, intron retention analysis also showed mild intron 70 retention in RMS cells which was confirmed by direct sequencing. Therefore, the identified retained introns might have arisen from malfunction of transcription regulation. From the results, intron retention could be a good focus of targeted therapy in RMS.
Next year, the applicants are going to search for candidate exonic/intronic splicing enhancers (ESE/ISE) by web based algorithms within the retained introns or nearby exons for AO-induced splicing analysis.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The applicants succeeded to identify intron retention of several dystrophin introns in three different Rhabdomyosarcoma cell lines and confirmed by direct sequencing. An indication that points of focus of RNA modulation and hence targeted therapy using antisense oligonucleotide was identified. These results were obtained as was expected from the proposal. Therefore, the work is progressing rather smoothly.
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| Strategy for Future Research Activity |
Based on the findings, a variety of antisense oligonucleotides (AO) will be tested for their efficacy as well as specificity to induce retained intron removal. The cell proliferation of rhabdomyosarcoma cells will also be evaluated with the different AOs.
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