Estrogen receptor-mediated miR-486-5p regulation of OLFM4 expression in ovarian cancer

Abstract

Estrogen signaling influences the development and progression of ovarian tumors, but the underlying mechanisms are not well understood. In a previous study we demonstrated that impairment of estrogen receptor alpha (ERα)-mediated olfactomedin 4 (OLFM4) expression promotes the malignant progression of endometrioid adenocarcinoma, and we identified OLFM4 as a potential target of miR-486-5p. In this study we investigated the role of OLFM4 in ovarian serous adenocarcinoma. Ovarian serous adenocarcinoma tissues had reduced OLFM4 expression. Expression of OLFM4 was positively correlated with ERα expression, and estrogen (E2) treatment in ovarian cancer cells induced OLFM4 expression in an ERα-dependent manner. In contrast to ERα, miR-486-5p levels were inversely correlated with OLFM4 expression in ovarian serous adenocarcinoma. Ovarian cancer cells transfected with miR-486-5p mimics showed decreased OLFM4 mRNA expression, and ovarian cancer cells treated with E2 showed reduced cellular miR-486-5p levels. OLFM4 knockdown enhanced proliferation, migration, and invasion by ovarian cancer cells. Low expression of OLFM4 was also associated with high tumor FIGO stage and poor tumor differentiation. These results suggest OLFM4 is downregulated by miR-486-5p, which contributes to ovarian cancer tumorigenesis. Conversely, estrogen receptor signaling downregulates miR-486-5p and upregulates OLFM4 expression, slowing the development and progression of ovarian cancer.

Keywords: OLFM4; estrogen; miR-486-5p; ovarian serous adenocarcinoma.

Figure 1. Expression of Olfactomedin 4 (OLFM4), estrogen receptor-a (ERα) and progesterone receptor (PR) in normal ovary (NO); ovarian serous cystadenoma (OSC); ovarian serous borderline tumor (OSBT); well-differentiated ovarian serous adenocarcinoma (WD-OSAC), moderately-differentiated ovarian serous adenocarcinoma (MD-OSAC) and poorly-differentiated ovarian serous adenocarcinoma (PD-OSAC) detected by immunohistochemistry

Haematoxylin-eosin (HE), Morphology of NO A. OSC E. OSBT I., and WD-OSAC M., MD-OSAC Q. and PD-OSAC (U) stained by haematoxylin-eosin. OLFM4, OLFM4 staining was undetectable in NO B.. Immunoreactivity of OLFM4 gradually increased from OSC F. OSBT J. to WD-OSAC N., and gradually decreased with lower degrees of differentiation in serous adenocarcinoma (N, R, V). ER, Immunoreactivity of ER gradually decreased from NO C. OSC G. OSBT K. to WD-OSAC O. ER staining was hardly detectable in MD-OSAC S. and PD-OSAC W. PR, Immunoreactivity of PR gradually decreased from NO D. OSC H. OSBT L., WD-OSAC P., MD-OSAC T. to PD-OSAC X.

Figure 2. Effects of OLFM4 on ovarian serous adenocarcinoma cells

A. Effects of OLFM4 on proliferation of HO8910-pm and SKOV3 measured by MTT (**P < 0.01). B. OLFM4 knockdown promoted migration of HO8910-pm cells by scratch wound assay. C. OLFM4 knockdown promoted invasion of HO8910-pm cells (*P < 0.05) by transwell invasion assay. D. Treatment of HO8910-pm cells with OLFM4 siRNA had no effect on cell apoptosis. E. Knockdown of OLFM4 in HO8910-pm cells transfected with specific siRNA resulted in decrease in G1 phase cells and increase in S phase cells (*P < 0.05).

Figure 3. Cumulative survival curve of patients with ovarian serous adenocarcinoma and expression of OLFM4

Figure 4. Expression of ERα

A. PR B. OLFM4 C. mRNAs, and OLFM4 protein D. in ovarian serous carcinoma cells, ***P < 0.001.

Figure 5. ERα-mediated regulation of OLFM4 expression in HO8910-pm cells

A. E2 induced the expression of OLFM4 and estrogen receptor antagonist ICI 182 780 attenuated the OLFM4 mRNA increase induced by E2. B. Knockdown of ERα expression with siRNA. C. Down-regulation of ERα with siRNA reduced the E2-induced expression of OLFM4. ***P < 0.001, **P < 0.01. NC: negative control.

Figure 6. Estrogen regulates the expression of miR-486-5p, which targets OLFM4

A. Comparison of miR-486-5p levels among formalin-fixed, paraffin-embedded (FFPE) tissues of normal ovary (NO), ovarian serous cystadenoma (OSC), ovarian serous borderline tumor (OSBT), and ovarian serous adenocarcinoma (OSAC) detected by IHC. B. miR-486-5p expression in frozen fresh ovarian serous adenocarcinoma and normal tissues detected by real-time PCR. C and N represent individual tissues of serous adenocarcinoma and normal ovary, respectively. C. OLFM4 expression in frozen fresh ovarian serous adenocarcinoma and normal tissues was detected by Western blot. D. Transfection of miR-486-5p mimics increased miR-486-5p levels in ovarian cancer cells. E. Increased miR-486-5p led to reduced OLFM4 mRNA levels. F. Treatment with estrogen (E2) resulted in decreased miR-486-5p levels. Treatment with estrogen receptor antagonist ICI 182 780 (ICI) or knockdown of ERα with ERα-specific siRNA attenuated E2-induced decrease of miR-486-5p levels in SKOV3 cells. G. Knockdown of ERα expression with ERα-specific siRNA in SKOV3 cells. NC: negative control, ***P < 0.001, **P < 0.01, and *P < 0.05.