Abstract
Steroid hormones govern the complex, cyclic changes of the endometrium, predominantly through their receptors. An interplay between steroid hormones and epigenetic mechanisms controls the dynamic endometrial gene regulation. Abnormalities in expression of genes and enzymes associated with steroid hormone signaling, contribute to a disturbed hormonal equilibrium. Limited evidence suggests the involvement of TET (Ten Eleven Translocation)-mediated DNA hydroxymethylation in endometrial cancer, with some data on the use of TET1 as a potential prognostic and diagnostic biomarker, however the mechanisms guiding it and its regulation remains unexplored. This study aims to explore the changes in the expressions of TETs and steroid hormone receptors in response to estrogen and progesterone in endometrial cancer cells. Gene expression was examined using real-time PCR and protein expression was quantified using fluorescent western blotting in endometrial cancer cell lines (AN3 and RL95-2). Results indicate that TET1 and TET3 gene and protein expression was cell-specific in cancer cell-lines. Protein expression of TET1 was downregulated in AN3 cells, while TET1 and TET3 expressions were both upregulated in RL95-2 cells in response to estrogen-progesterone. Further, a decreased AR expression in AN3 cells and an increased ERα and ERβ protein expressions in RL95-2 cells was seen in response to estrogen-progesterone. PR gene and protein expression was absent from both cancer cell-lines. Overall, results imply that expressions of steroid hormones, steroid-hormone receptors and TETs are co-regulated in endometrial cancer-cells. Further studies are needed to interpret how these mechanisms fit in with DNMTs and DNA methylation in regulating endometrial biology. Understanding the role of TETs and hydroxymethylation in steroid hormone receptor regulation is crucial to comprehend how these mechanisms work together in a broader context of epigenetics in the endometrium and its pathologies.
Highlights
A two-way communication between epigenetic mechanisms and steroid hormones is crucial for the healthy functioning of the endometrium
This study is aimed at mimicking the hormonal influences seen during the menstrual cycle in vitro, to explore the mechanisms involved in the regulation of ten-eleven translocation (TET) and Abbreviations: 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; androgen receptors (AR), Androgen Receptor; AREs, Androgen Responsive Elements; charcoal stripped FBS (CS-FBS), Charcoal stripped FBS; C treatment, Control; DNMTs, DNA Methyltransferases; E treatment, Estradiol treatment; EP treatment, Estrogen + progesterone treatment; Era, Estrogen receptor alpha; ERb, Estrogen receptor beta; estrogen receptor (ER), Estrogen receptors; estrogen related receptor alpha (ERRa), Estrogen related receptor alpha; progesterone receptor (PR), Progesterone receptor; TDG, thymine DNA glycosylase; TET, Ten-eleven translocation
Protein expression for steroid hormone receptors revealed no significant changes in ERb expression
Summary
A two-way communication between epigenetic mechanisms and steroid hormones is crucial for the healthy functioning of the endometrium. Transcriptional regulation of steroid hormone receptors in the endometrium is partly controlled by epigenetic factors like DNA methylation and hydroxymethylation [1–5]. DNA methylation yields 5-methylcytosine (5mC), making for one of the most important forms of epigenetic modification in the mammalian DNA [6]. The modification of DNA from 5C (5-Cytosine) to 5mC can be actively or passively reversed via the process of DNA de-methylation. 5hmC is identified as an independent epigenetic modification that can alter gene expression and might be important in epigenetic reprogramming [8]. The active de-methylation process is catalysed by ten-eleven translocation (TET) enzymes, making them an essential component in epigenetic machinery. DNA methylation is known to be involved in maintaining successful steroid hormone signaling by regulating steroid hormone receptors [16]. Estrogen and progesterone can influence mRNA and protein expression of DNA Methyltransferases (DNMTs), thereby affecting methylation patterns [17–19]
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