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. 2022 Mar 11:12:763464.
doi: 10.3389/fonc.2022.763464. eCollection 2022.

Differential Expression of Steroid Hormone Receptors and Ten Eleven Translocation Proteins in Endometrial Cancer Cells

Affiliations

Differential Expression of Steroid Hormone Receptors and Ten Eleven Translocation Proteins in Endometrial Cancer Cells

Vishakha Mahajan et al. Front Oncol. .

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.

Keywords: DNA hydroxymethylation (5hmC); endometrial cancer cells; gene expression; steroid hormones and receptors; ten eleven translocation (TET proteins).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Relative TET1 TET2 and TET3 mRNA expression in response to steroid hormone treatment in AN3 cells. The y-axis shows the fold change of mRNA levels following different treatments compared with control, all results corrected against geo-mean expressions of three reference genes - YWHAZ, RPLO and RPL13a. The x-axis shows different treatment groups. E24 = 24h Estrogen; EP24, EP48 and EP72 = both Estrogen + Progesterone for 24, 48 and 72h. Data are presented as mean ± SEM *p < 0.05. P ≤ 0.1 was considered as approaching significance. The experimental setup included three independent sets of cell culture experiments (n = 3) and triplicates of each sample for the RT-PCR.
Figure 2
Figure 2
TET protein expression in response to different steroid hormone treatments in AN3 cells. A representative blot image for the particular weight band is shown next to the graph. The y-axis shows the fold change of protein levels following different treatments compared to control and x-axis shows the different treatment groups. E24, 24h Estrogen; EP24, EP48 and EP72 = both Estrogen + Progesterone for 24, 48 and 72h. Data are presented as mean ± SEM, *p < 0.05 (P ≤ 0.1 is considered as approaching significance). The experimental setup included three independent sets of cell culture experiments (n = 3) with three technical replicates for each sample.
Figure 3
Figure 3
Steroid hormone protein expression in response to different steroid hormone treatments in AN3 cells. A representative blot image for the particular weight band is shown next to the graph. The y-axis shows the fold change of protein levels following different treatments compared to control and x-axis shows the different treatment groups. E24 = 24h Estrogen; EP24, EP48 and EP72 = both Estrogen + Progesterone for 24, 48 and 72h. Data are presented as mean ± SEM. P ≤ 0.1 is considered as approaching significance. The experimental setup included three independent sets of cell culture experiments (n =3) with three technical replicates for each sample.
Figure 4
Figure 4
Relative TET and ERb mRNA expression in response to different steroid hormone treatments in RL95-2 cells. The y-axis shows the fold change of mRNA levels following different treatments compared with control, all results corrected against geo-mean expressions of three reference genes - YWHAZ, RPLO and RPL13a. The x-axis shows different treatment groups E24 = 24h Estrogen; EP24, EP48 and EP72 = both Estrogen + Progesterone for 24, 48 and 72h. Data are presented as mean ± SEM; ***P < 0.001; P ≤ 0.1 was considered as approaching significance. One way ANOVA test revealed significant variations in TET and ERb expression across treatments (****P<0.0001). The experimental setup included three independent sets of cell culture experiments (n =3) and triplicates of each sample for the RT-PCR.
Figure 5
Figure 5
TET protein expression in response to different steroid hormone treatments in RL95-2 cells. A representative blot image for the particular weight band is shown next to the graph. The y-axis shows the fold change of protein levels following different treatments compared to control and x-axis shows the different treatment groups. E24 = 24h Estrogen; EP24, EP48 and EP72 = both Estrogen + Progesterone for 24, 48 and 72h. Data are presented as mean ± SEM, *p < 0.05, P ≤ 0.1 is considered as approaching significance. The experimental setup included three independent sets of cell culture experiments (n =3) with three technical replicates for each sample.
Figure 6
Figure 6
Steroid hormone protein expression in response to different steroid hormone treatments in RL95-2 cells. A representative blot image for the particular weight band is shown next to the graph. The y-axis shows the fold change of protein levels following different treatments compared to control and x-axis shows the different treatment groups. E24 = 24h Estrogen; EP24, EP48 and EP72 = both Estrogen + Progesterone for 24, 48 and 72h. Data are presented as mean ± SEM, *p < 0.05, P ≤ 0.1 is considered as approaching significance. The experimental setup included three independent sets of cell culture experiments (n = 3) with three technical replicates for each sample.

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