Rason, Nursyuhana
(2014)
Study on regulation of VGSCS expression by REST
using AZA in MCF-7 breast cancer cell line.
Project Report.
Universiti Sains Malaysia.
(Submitted)
Abstract
The Repressor Element 1-Silencing Transcription factor (REST) mediates the repression of
several neuronal genes in non-neuronal cells. REST represses its target gene through
histone deacetylation, chromatin remodeling and methylation. Loss of DNA methylation
has been reported as an early event in tumorigenesis and could possibly explained the
increased expression of neuronal genes including voltage-gated sodium channels (VGSCs)
in cancer cells. The question remain, is REST regulates VGSC expression in breast cancer?
In this study, a DNA methylation inhibitor 5-azacytidine (AZA) was used to inhibit REST
function and test its effects on proliferation and the expression of REST common target
genes and VGSCs in the weakly metastatic breast cancer cells, MCF-7. MTT assay was
carried out to determine the effect of AZA treatment on cell growth at different
concentration, 100 pM - 1 mM for 24, 48 and 72 h. Total RNA extraction, cDNA
synthesis, PCRs, gel electrophoresis and image analysis were conducted to investigate the
effects of AZA on the gene expression of synaptophysin (SYP), chromagranin A (CHGA),
both REST common target genes and VGSCs (Nav1.5 and nNav1.5). Results showed that
AZA caused a dose-dependent decreased on the viability of MCF-7 cells where the effect was prominent at highest concentration, 1 mM after 72 h of treatment. REST common target genes, SYP and CHGA had an increased pattern in expression after AZA treatment.
Interestingly, an increased expression pattern of Nav1.5 and nNav1.5 was also observed, though the increments of expression of these genes were not significant. Herein, a possibility that Nav1.5 and nNav1.5 in breast cancer cells are REST target gene could be
postulated, though further works are needed to confirm the interaction.
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