The Cell-Free Expression of MiR200 Family Members Correlates with Estrogen Sensitivity in Human Epithelial Ovarian Cells

Abstract

Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.

Comparative Analysis of Cell-Free miR-205-5p, let-7f-5p, and miR-483-5p Expression in Ovarian Cell Cultures and Plasma Samples of Patients with Ovarian Cancer

“…In our previous studies, we identified several cell-free miRNAs (e.g., miR-200 family members and miR-203a) as candidate biomarkers with good diagnostic parameters in ovarian cancer [9,10]. We also confirmed high cell-free miR-200s expression in estrogen sensitive epithelial ovarian cell cultures [11]. Here, we aimed to identify additional new miRNAs that might be used as future biomarkers in liquid biopsy.…”

“…We applied two cell lines that differed in the expression of Estrogen Receptor α (ERα); that receptor highly determined the expression of miRNAs in our previous studies [11]. The PEO1 highly expresses the Estrogen Receptor α (ERα) and proved to be estrogen sensitive in our previous studies [11]. The other cell line used was A2780 which does not express ERα and, as a result, does not respond to estrogen exposure, as it was shown previously [11].…”

“…When the intracellular expression of miRNAs was studied, total RNA, including small non-coding RNAs, were isolated by the miRNeasy Kit (Qiagen, Hilden, Germany) from the cell lysates according to the manufacturer's instructions. When cell-free miRNAs secreted by the cell cultures were studied, the supernatant of cultures was used as a source for miRNA isolation using miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, as previously described [11].…”

“…The expression of miR-483-5p, miR-205-5p, and let-7f-5p was determined by the miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) using miRNA specific miScript primer Assays (Qiagen, Hilden, Germany) as previously described [9,11]. MiR-103-3p was used as a housekeeping miRNA that proved to be an applicable reference in our previous studies [9][10][11]. Relative expression level was determined relative to miR-103-3p expression, where the relative expression level of a specific miRNA is equivalent with 2 −∆Ct .…”

“…In the beginning of our work, the basal expression level of miR-483-5p, miR-205-5p, and let-7f-5p was determined in epithelial ovarian cell lines. We applied two cell lines that differed in the expression of Estrogen Receptor α (ERα); that receptor highly determined the expression of miRNAs in our previous studies [11]. The PEO1 highly expresses the Estrogen Receptor α (ERα) and proved to be estrogen sensitive in our previous studies [11].…”