Kamarulzaman, Nur Sabrina
(2020)
Elucidating the function of repressor element silencing transcription factor in human breast cancer and its relation with voltage-gated sodium channels-mediated metastasis.
Masters thesis, Universiti Sains Malaysia.
Abstract
Voltage-gated sodium channels (VGSCs), particularly isoform Nav1.5 and
its neonatal splice variant, nNav1.5, is found to be highly upregulated in human
breast cancer and its expression/activity associates with strong metastatic potential.
Whilst repressor element silencing transcription factor (REST) was discovered as
tumour suppressor in various type of carcinomas in which loss/lack of its expression
has been linked to aggressive phenotype. The overall aim of this study was to
investigate the role of REST in regulating Nav1.5 and nNav1.5 expression in human
breast cancer cells. Real-time PCR and Western blotting were conducted to compare
the mRNA and protein expression levels of target molecules (Nav1.5, nNav1.5,
REST, HDAC1, HDAC2, HDAC3, MMP2, N-cadherin), respectively, between the
non-cancerous breast epithelial cell line (MCF-10A), the less aggressive human
breast cancer cell line (MCF-7) and the highly aggressive human breast cancer cell
line (MDA-MB-231). The possible REST binding sites on Nav1.5 promoter
sequence was predicted using online software JASPAR2018. Since MCF-7 cells has
been reported to expressed higher REST expression, siRNA-mediated REST
knockdown and treatment using histone deacetylase (HDAC) inhibitor, trichostatin
A (TSA) was performed only on MCF-7 cells. Cell growth and metastatic
behaviours of the cells were also assessed by functional assays (MTT, lateral
motility and migration assays). REST mRNA was detected in all three cell lines with
the highest expression in MCF-7 cells (p<0.05). Similarly, REST protein expression
was also detected in all three cell lines, with the highest expression in MCF-7 cells
(p<0.05). The mRNA expression of Nav1.5 (p<0.01) and nNav1.5 (p<0.05) was
higher in MDA-MB-231 cells compared to MCF-7 cells and not detected in MCF-
10A cells. Correspondingly, Nav1.5 protein expression was also higher in MDAMB-
231 cells compared to MCF-7 cells (p<0.05). Twelve REST binding sites in
Nav1.5 promoter were predicted by JASPAR2018. Although mRNA and protein
expression of REST were successfully knockdowned (p<0.05), however, no
significant change was observed on Nav1.5 and nNav1.5 mRNA level. Additionally,
although only HDAC2 mRNA expression was significantly higher in MCF-7 cells
compared to MDA-MB-231 cells (p<0.05) (from the basal comparison analysis),
instead, in the MCF-7-REST knockdown cells, HDAC1 mRNA expression was
significantly reduced (p<0.05). In the TSA treated MCF-7 cells, HDAC2 mRNA
level was significantly reduced (at 1000 and 10 000 ng/ml, p<0.001), similarly,
REST mRNA expression was significantly reduced (at 100 (p<0.05), 1000
(p<0.001) and 10 000 (p<0.0001) ng/ml)). Interestingly, TSA significantly enhanced
mRNA expression of Nav1.5 (at 1000 (p<0.05) and 10 000 (p<0.01) ng/ml) and
nNav1.5 (at 10 000 ng/ml, p<0.01). This was followed by enhanced cell migration
(at 1000 and 10 000 ng/ml, p<0.05), which was confirmed by significant
upregulation of two metastasis-related genes, MMP2 (p<0.01) and N-cadherin
(p<0.05). In conclusion, REST regulates Nav1.5/nNav1.5 expression via inhibition
by HDAC2 in the less aggressive breast cancer cells (which did not occur in
aggressive breast cancer due to lack of REST), providing a revelation that
Nav1.5/nNav1.5 expression in breast cancer could be regulated by epigenetics.
| Item Type: |
Thesis
(Masters)
|
| Uncontrolled Keywords: |
Breast neoplasms |
| Subjects: |
R Medicine |
| Divisions: |
UNSPECIFIED |
| Depositing User: |
Mr Abdul Hadi Mohammad
|
| Date Deposited: |
18 Jul 2021 02:49 |
| Last Modified: |
18 Jul 2021 02:49 |
| URI: |
http://eprints.usm.my/id/eprint/49466 |
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