qPCR validation of in vivo diagnostic importance and regulation by estrogen for CHRNA5 isoform expression in breast cancer
Author
Özdemir, Emine Sıla
Advisor
Konu, Özlen
Date
2014Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
Breast cancer has multiple molecular subtypes; normal-like, basal-like, luminal A,
luminal B and HER2 positive depending on receptor status of tumor cells. Cancer
therapy is tailored according to the type of cancer; hence finding new diagnostic
markers is important to decide on the best treatment approach. Cholinergic nicotinic
receptor alpha 5 (CHRNA5) is one of the subunits of nicotinic acetylcholine
receptors with significant roles in addiction and cancer. In the present study,
CHRNA5 has been validated as an estrogen and/or Estrogen receptor (ER)
modulated nicotinic acetylcholine receptor by qPCR in in vitro and in vivo in breast
cancer samples. CHRNA5 isoform expression was measured using in vitro cell
culture studies in which ER- and ER+ cell lines treated with different doses of
estradiol (E2); MCF7 cell line was exposed to long-term E2 depletion, in another
experiment it was treated with tamoxifen (4-OHT), an ER antagonist, and with or
without E2. We found that all CHRNA5 isoforms exhibited increased expression in
response to E2 dose-dependently in the ER+ MCF7 cell line while in the ER- MDAMB-231
cell line CHRNA5 isoform expression response was variable in direction
and magnitude. CHRNA5 isoform expression in general steadily decreased in ER+
cell line MCF7 after 4-OHT treatment. After six months of E2 depletion, ER+ MCF7
cell line had increased CHRNA5_v3 isoform and ESR1 (ER gene) mRNA expression. In vivo, a human breast cancer cDNA panel was scanned with specially
designed primers with qPCR using a custom-written GUI in MATLAB. It was found
that CHRNA5, showing a statistically significant difference between normal and
tumor cDNA, was a good candidate gene in diagnosis of breast cancer. CHRNA_v3
was able to distinguish between ER+ vs ER- breast tumor samples. We also
addressed whether CHRNA5 isoforms exhibited differences in distinguishing tumor
stage, and HER2 status. Our findings showed that expression of CHRNA5 isoforms
were correlated with each other and regulated by E2 in breast cancer depending on
ER receptor status.