Gene expression analysis of oestrogen receptors and atypical chemokine receptors in response to 17-β oestradiol in MCF-7
Creators: name: Talashi, Shruti
Publisher: name: Zenodo
Year: 2024
Subjects: E2-17-Estradiol
ER- Oestrogen receptor alpha gene
ER – Oestrogen receptor beta gene
TME – Tumour microenvironment
ACKRs – Atypical Chemokine receptors (ACKR2 gene, ACKR3 gene &amp; ACKR4 gene)
RT-qPCR – Real time quantitative polymerase chain reaction;
HKGs- Housekeeping genes
18S rRNA – 18S ribosomal ribonucleic acid gene
HPRT1- Hypoxanthine Phosphoribosyltransferase 1 gene
TBP- Tata Box binding gene ;
TOP1- Topoisomerase 1
DMSO – Dimethyl Sulfoxide
mRNA – messenger ribonucleic acid

description: 17-Estradiol (E2) is the most potent form of oestrogen, that binds efficiently to oestrogen receptors type i.e. Oestrogen receptor-alpha (ER) and oestrogen receptor-beta (ER) to regulate the cell growth in the tissues. E2 is crucial for the breast development, yet a high level of E2 has been associated with cancer. Inflammation as a cancer hallmark, involve chemokine's which are signalling molecules that attract immune cells to promote cancer spread or metastasis. Interestingly, in cancer biology research, E2 has shown ability to inhibit chemokine production in tumour microenvironment (TME), although the mechanism remains unclear. There are chemokine receptors as atypical chemokine receptors (ACKRs) which are referred to as anti-inflammatory G-protein independent transmembrane proteins that act like "scavengers," removing chemokine's and dampening inflammation. Atypical chemokine receptor type 2 (ACKR2) potentially lowers inflammation & is established as essential gene for the mammary gland development. ACKR 2 is a gene essential for development and it also act as a tumour suppressors. Unlike ACKR 2, ACKR 3 & ACKR 4 are associated with cancer progression. A higher E2 level is found around breast cancer tissues and E2 has the ability to lower chemokine production in cancer environment. E2 can be considered as preventive to cancer from spreading and since ACKRs being expressed in breast cancer. This research delves into this paradox by conduct experiment to investigate the higher E2 effect on ACKRs gene modulation using a hormone responsive breast cancer model. 

In-vitro MCF-7 cell line used as experimental model since it is hormone responsive and most studied breast cancer cell line. For gene expression analysis, RT-qPCRtechnique been optimised to study gene modulation of target gene of interest as ERand ACKRs when exposed to higher E2 levels. Use of published primers for ER gene isoforms led to multiple (non-specific binding) PCR products. Experiment has been designed to make selection of normalization gene for use in RT-qPCR from few housekeeping gene (HKGs) that had been used in gene expression study before 18S rRNA, HPRT1, TBP while introducing TOP1 gene, for which primers were designed in-house. Using 'Multialign' and 'Oligocalci' online bioinformatics tool, a new primer was designed for ER gene and called as ER_iso primer.TOP1 as HKG had been chosen as best normalization gene for RT-qPCR assay. E2 (1nM, 100nM, 1M) & their vehicle control (DMSO ≤ 0.1% v/v) were used to prepare E2 stimulated samples for gene expression study at two points 6 hours(H) & 18H. Relative gene expression were calculated applying Livak's ∆∆ Cq comparative method and for statistical analysis non-parametric Mann-Whitney's U-test had been used. Experimental analysis shown one-fold down regulation of mRNA gene expression of ER  & two-fold down regulation of mRNA gene expression ACKR 2, ACKR 3 & ACKR 4 at transcriptional (m-RNA) level. Statistical analysis support this fact as there lies a significant differences (p < 0.05) between E2 treated and vehicle control groups at 18H with a confidence interval 95%, hence E2 modulate the mRNA gene expression of ACKRs antiinflammatory mediator.These findings highlight the seemingly contradictory roles of E2. Since it has been considered to fuel tumour growth, it might also have anti-inflammatory properties through a pathway that involve ACKRs gene. Hence it is deemed to further research into the interplay of ERs & ACKRs that could be valuable in finding early biomarkers in breast cancer screening or in developing novel cancer therapies.