In-Vitro Cell Based Study Of Smn2 Expression Upon Exposure With Histone Deacetylase Inhibitors And Polyphenols As Potential Therapy For Spinal Muscular Atrophy
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Date
2015-10
Authors
MOHSENI, JAFAR
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Autosomal recessive SMA is the second most common inherited disease, leading to early infancy death. The SMN1 mutations are leading cause of SMA. However, increasing expression of SMN2 has been exploited as therapeutic target for Spinal Muscular Atrophy (SMA). Several Histone Deacetylase Inhibitors (HDACIs) are known to increase SMN2 expression level. This study aimed to elucidate the effects of two hydroxamate-based HDACIs, SAHA and Dacinostat, and one SIRT1 activator, SRT1720, on the SMN2 expression, CpG Islands (CGI) methylation and SMN protein levels in fibroblasts taken from SMA Type I and Type II patients.
Toxicity assay/dosage determination was carried out using trypan blue exclusion technique. Each cell type was treated with serial concentrations of Dacinostat, SRT1720 and SAHA and their combinations for 48 hr. Quantification of Overall-SMN2 expression (Overall-SMN2) and SMN2 exon 7 inclusion (E7-SMN2) was subsequently done using QuantiGene® 2.0 Plex assay (Affymetrix). SMN2 CGIs methylation level was investigated using MS-HRM (Methylation-Specific High Resolution Melting) on bisulfite treated DNA. SMN protein was quantified using ELISA method using SMN protein assay kit (Enzo Life Science, USA). Methodologically, exclusive increase in E7-
SMN2 (without increase in Overall-SMN2) should result in the decrease of SMN2 transcripts that lack exon 7 (Δ7-SMN2). Δ7-SMN2 quantification in this study was inferred from the difference between Overall-SMN2 and E7-SMN2. The p-values were considered statistically significant if they are less than 0.05 for comparison between untreated and treated cells.
It was found that the levels of SMN2 gene expression (Overall-SMN2 and E7-SMN2) and SMN protein level were significantly increased in 10 μM SAHA-treated Type I and Type II cells compared to untreated cells (Mock). The mean methylation levels in CGIs of SMN2 CpG Islands region showed significantly lower levels of methylation as well. Accordingly, the level of Overall-SMN2 and E7-SMN2 transcript increase were also significant in 32 nM Dacinostat-treated fibroblasts type I and type II cells as compared to untreated fibroblasts (Mock). The transcript increase induced by Dacinostat led to more increase of SMN protein compared to SAHA in Type I cells (2.54 + 1.57 fold). The Δ7-SMN2 transcript in type I and type II cells were noted to be decreased, although not significant. Moreover, methylation levels in CGIs of SMN2 CpG Islands region showed statistically lower percentage in at 5% level of significance.
SMN2 gene expression (Overall-SMN2 and E7-SMN2) was remarkably increased upon exposure to SRT1720. While, the mean CGIs methylation percentage and SMN protein level alteration were decreased modestly. SAHA-Dacinostat combination increased SMN2 expression in Type I, but not Type II cells. SRT1720-Dacinostat combination increase Overall-SMN2 and E7-SMN2 transcripts nearly double the increase induced by individual compounds. SRT1720-SAHA combination resulted in lower increase than
that induced by SAHA alone but higher increase than that induced by SRT1720 alone. Furthermore, SMN protein was noted to be increased and CGIs was more demethylated in treated cells.
In conclusion, SMN2 expression (Overall-SMN2 and E7-SMN2), SMN protein level and methylation level of CGIs were significantly altered upon exposure to SAHA, Dacinostat and SRT1720-Dacinostat combination in SMA fibroblast Type I and Type II. This is the first report about Dacinostat and SRT1720 effect on SMN2 modulation.
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Keywords
Autosomal recessive SMA , common inherited disease.