Changes in the Transcriptome of the Human Endometrial Ishikawa Cancer Cell Line Induced by Estrogen, Progesterone, Tamoxifen, and Mifepristone (RU486) as Detected by RNA-Sequencing

Abstract

BackgroundEstrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.Methodology/Principal findingsGene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1. ConclusionsSignificant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.

Effect of single post-ovulatory administration of mifepristone (RU486) on transcript profile during the receptive period in human endometrium

“…P 4 has been shown to completely inhibit estrogen-induced epithelial cell proliferation and DNA synthesis which is in line with the functional clustering related with 'proliferation.' Moreover, a functional analysis of mifepristone responsive genes in Ishikawa cells identified a signaling pathway associated with adhesion and cellto-cell interactions (Tamm-Rosenstein et al 2013), which is in line with the term 'cell adhesion' we found. In relation to the over-represented TBFS analysis in the promoter regions of endometrial genes regulated by mifepristone, it is interesting that the cognate sequence for PGR was not over represented, suggesting that the transcriptional regulation exerted by the PGR is presumably mostly indirect.…”

Polycomb subunit BMI1 determines uterine progesterone responsiveness essential for normal embryo implantation

“…By contrast, we noted comparable endometrial expression levels of PR and E6AP in both groups (Figure 7, A, D, and E). These aberrantly lower expression levels of BMI1, but not PR and E6AP, in failed pregnancy were well associated with reduced expression levels of PR-target genes including FOSL2, JUN, TGFB1, and IRS (18,19,23) (Figure 7F), indicating a close correlation between BMI1 and P4-PR nuclear signaling in human endometrium during early pregnancy.…”

“…As shown in Supplemental Figure 8, we first generated a BMI1-null mutant Ishikawa cell line utilizing the CRISPR/Cas9 knockout strategy. It is interesting to note that null mutation of BMI1 in Ishikawa cells significantly reduced the expression levels of PR target genes, such as TGFB1 and NPAS2 (18,19) (Figure 4B) and hampered PR transcriptional activity as well when assessed by a P4 response element (PRE)-luciferase reporter assay upon P4 challenge ( Figure 4C). In line with the in vivo observation that supplementation of exogenous P4 can partially correct implantation defects in Bmi1…”

Pigment Epithelium–Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia

“…As demonstrated by gene array profiling, these genes are related to cellular processes of DNA replication, cell-cycle progression, and cellular organization (12). Yet, the exact molecular mechanism by which tamoxifen exerts adverse effects on the uterus remains to be elucidated.…”