Estrogenic transmembrane receptor of GPR30 mediates invasion and carcinogenesis by endometrial cancer cell line RL95-2

Abstract

Purpose: The mechanisms underlying the effects of estrogen on endometrial cancer remain undefined. Although the classical mechanism of the action of estrogen involves binding to the estrogen receptors α and β, and transduction of the signal into the cell, G protein-coupled receptor (GPR) 30 has been shown to mediate nongenomic estrogen signaling. The goal of this study was to determine the role of GPR30 signal in the basic process such as invasion and carcinogenesis of endometrial cancer.

Methods: We downregulated the expression of GPR30 in endometrial cancer cell line RL95-2 by transfection with shGPR30-pGFP-V-RS, a GPR30 antisense expression vector. The cells were then subjected to an MTT assay and a Transwell(®) migration assay. And an animal model was also used to investigate the influence of downregulation of GPR30 on oncogenesis.

Results: Downregulation of GPR30 led to reduced growth and invasion by cells treated with 17β-estradiol. And the capacity of transfected RL95-2 cells to promote tumorigenesis was weakened in vivo.

Conclusions: Our data suggest that, for the endometrial cancer cell line RL95-2, GPR30 plays important roles in mediating the proliferative and invasive effects of estrogen and in tumorigenesis.

Fig. 1

The pGFP-V-RS plasmid vector pattern and interferential efficiency of ShGPR30-pGFP-V-RS vectors. a The pGFP-V-RS plasmid vector was created with an integrated turboGFP element to readily verify transfection efficiency. It also incorporates both a kanamycin and puromycin resistance elements for greater selection capabilities. The shRNA expression cassette consists of a 29-nt GPR30-specific sequence, a 7-nt loop, and another 29-nt reverse complementary sequence, all under the control of the human U6 promoter. A termination sequence (TTTTTT) is located immediately downstream of the second 29-nt reverse complementary sequence to terminate the transcription by RNA Pol III. b The RNA expression levels of RL95-2 cells transfected with each of four different ShGPR30-pGFP-V-RS vectors were determined using real-time RT-PCR. RNA expression of RL95-2 cells transfected with shiv-pGFP-V-RS (negative control) was set at 1, from which the RNA expression of the other cells was calculated. c RL95-2 cells were visualized by fluorescence microscopy after stable transfection with pGFP-V-RS(b). a Cells transfected with ShGPR30-pGFP-V-RS under white light. b Cells transfected with ShGPR30-pGFP-V-RS under fluorescence. c Cells transfected with shiv-pGFP-V-RS (negative control) under white light. d Cells transfected with shiv-pGFP-V-RS (negative control) under fluorescence. Magnification, (a, b) ×200; (c, d) ×100

Fig. 2

G protein-coupled receptor 30 via the MEK/ERK mitogen-activated protein kinase (MAPK) pathway stimulates proliferation of an endometrial cancer cell line. a RL95-2 cells were treated with 17β-estradiol (E2, 10⁻⁹ ~ 10⁻⁵M) or G1 (10⁻⁹ ~ 10⁻⁵ M) for 24 h in medium containing 5% charcoal-stripped fetal bovine serum then analyzed on the indicated cultured times. ^# P < 0.05 versus the other five control groups, ^* P < 0.05 versus the other five RNAi groups; b RL95-2 cells were treated with E2 (10⁻⁸M) and G1(10⁻⁸M) for 24 and 48 h. ^# P < 0.05 versus vehicle 24 h; ^* P < 0.05 versus vehicle 48 h. c, d: RL95-2 cells were treated with E2 (10⁻⁸ M) or G1 (10⁻⁸ M) with or without pertussis toxin (PTX) (200 ng/mL) or U0126 (30 μM) in medium containing 5% charcoal-stripped fetal bovine serum. Cells were further cultured for 24 h before evaluation of cell proliferation. Vehicle: 0.1% dimethyl sulfoxide/phenol red-free and serum-free Dulbecco’s modified Eagle’s medium/F12. Negative control: cells transfected with HuSH 29-mer noneffective against enhanced green fluorescent protein (shiv-pGFP-V-RS vector). RNAi: cells transfected with HuSH 29-mer short hairpin RNA against GPR30 in pGFP-V-RS vector (ShGPR30-pGFP-V-RS vector). ^* P < 0.05; ^Δ P > 0.05; ^& P < 0.05 versus the negative control. Medium was refreshed and treatments were renewed every 2 days. Each data point is the mean ± SD of three independent experiments

Fig. 3

G protein-coupled receptor 30 stimulates invasion by an endometrial cancer cell line via the MEK/ERK mitogen-activated protein kinase (MAPK) pathway. RL95-2 cells stably transfected with shiv-pGFP-V-RS (negative control; a–g) or ShGPR30-pGFP-V-RS (h–n) were treated as follows. a, h Control group (vehicle 0.1% dimethyl sulfoxide/phenol-free Dulbecco’s modified Eagle’s medium/F12; b, i 17β-estradiol (E2) (10⁻⁷ M); c, j E2 (10⁻⁷ M) with U0126 (30 μM); d, k E2 (10⁻⁷ M) with pertussis toxin (PTX) (200 ng/mL); e, l G1 (10⁻⁸ M); f, m G1 (10⁻⁸ M) with U0126 (30 μM); g, n G1 (10⁻⁸ M) with PTX (200 ng/mL). Cell invasion was then assessed and photomicrographs taken. Quantitation (o, p) and representative photomicrographs (a–n) show inhibition of invasion by RL95-2 cells transfected with ShGPR30-pGFP-V-RS vector (RNAi) compared with the negative control (shiv-pGFP-V-RS). Data are expressed as the mean ± SD. ^* P < 0.05 compared with the negative control

Fig. 4

G protein-coupled receptor 30 (GPR30) signaling in endometrial carcinoma cells enhances their tumorigenic capacity. a Volume of tumors produced by RL95-2 cells stably transfected with shiv-pGFP-V-RS (control) or ShGPR30-pGFP-V-RS (GPR30 RNAi) in nude mice over 56 days and b tumors taken from nude mice 56 days after inoculation of RL95-2 cells. Data are expressed as the mean ± SD