| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所 === 97 === Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions/mutations in the telomeric copy of the survival motor neuron gene (SMN1) on chromosome 5q13. A second centromeric copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to the alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is unstable and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. We screened for hundreds of compounds by using NSC34 motor neuron cell lines with SMN2 minigene (exon 6-exon 8)-luciferase system, and had identified the four compounds: oxa-32, oxa-19, CH-15 and #91 that significantly increased the luciferase activity. We also showed that oxa-32 and #91 promotes SMN2 exon 7 inclusion in SMA patient-derived cell lines, but no significant effect for oxa-19, CH-15. However, oxa-32 has no significant effect on SMN protein expression. To explore the mechanism of drug effect, we analyzed if #91 affected SR and hnRNP protein expression in SMA cells. We found that #91 down-regulates hnRNPA1. According to the models of SMN2 exon 7 splicing, we suggest that the compound #91 enhances SMN2 exon 7 inclusion by suppressing hnRNPA1 expression. In addition, we constructed a new system for drug screening. The new system was built in NSC34 motor neuron cell containing SMN2 promoter and SMN2 minigene-luciferase. By using this system, we can screen drugs that promoting the exon 7 inclusion in SMN2 mRNA and/or increasing SMN2 gene transcription at the same time. We screened for more than four hundred compounds and identified three compounds, WTC1467q, CYL840h, and Bifido 15476, that increase luciferase activity. We detected RNA and protein expression to figure out the effect of these compounds is to promote the exon 7 inclusion in SMN2 mRNA or increase SMN2 gene transcription. The results showed that WTC1467q and CYL840h had no significant effects of promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription. Bifido 15476 has the ability to increase SMN FL mRNA, but causes no significant change in SMN protein expression. We will confirm the potential of Bifido 15476 as a therapeutic drug. We will also screen more compounds and investigate the mechanism of those compounds that can promote the exon 7 inclusion in SMN2 mRNA and/or increase SMN2 gene transcription.
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