The human NSCLC cell lines (A549, LTEP-a2 and H358) and human normal bronchial epithelial cells (HBE, and NC004) were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). They were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37̊C in a humidified atmosphere of 5% CO₂. Cells were cultured in a phenol-red free medium supplemented with 5% charcoal stripped FBS (Sangon Biotech Co., Ltd, Shanghai, China) for at least 18 h prior to transfection. The ERβ2-specific mouse anti-human monoclonal antibody (57/3; Serotec, Kidlington, UK) and p38MAPK-specific rabbit anti-human monoclonal antibody (4511; Cell Signaling Technology Inc., Danvers, MA USA) were conserved in our laboratory. The p38MAPK inhibitor (SB203580) was purchased from Calbiochem (San Diego, CA, USA).

ERβ2-, p38MAPK- and IL-12Rβ2- (9,14) expressing plasmids (p3xFlag-ERβ2, p3XFlag-p38MAPK and pXJ40-Myc-IL-12Rβ2) were produced through the ligation of polymerase chain reaction (PCR)-generated inserts into p3xFlag-CMV-7.1–2 or pXJ40-Myc-SOX4, as appropriate. The small hairpin (sh) IL-12Rβ2 plasmids were purchased from the national RNAi core Facility located at the Institute of Molecular Biology/Genomic Research Center (Academia Sinica, Taipei, China).

The purified p3xFlag- ERβ2/p38MAPK and p XJ40-Myc-IL-12Rβ2 plasmids were transfected into 70% confluent A549, LTEP-a2 and H358 cells, using Lipofectamine^® 2000 (Invitrogen Life Technologies) reagents in a total volume of 1 ml of Opti-MEM (Invitrogen Life Technologies), as described in a previous study (23). A549, LTEP-a2 and H358 cells were co-transfected with 1 mg p3xFlag-p38MAPK, pXJ40-Myc-IL-12Rβ2, p3xFlag-empty or pXJ40-Myc-empty plasmids.

After transfection (12 h), cells were seeded in triplicate at a density of 5×10³ cells per 96-well plates. The following day, cells were treated with 10 mM estrogen (E2) dissolved in ethanol. At 24, 48 and 72 h after E2 treatment, 10 μl of a modified 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide solution (MTT; Dojindo, Kumamoto, Japan) was added to the culture and reaction mixtures were incubated at 37°C for 2 h. In order to detect migration, cells were placed in transwell chambers (Forma™; Thermo Fisher Scientific, Waltham, MA, USA) at 2×10⁴ cells/well. The lower transwell chamber contained 10% FBS as a chemoattractant. For the invasion assay, the bottom of the culture inserts (8-mm pores) were coated with 30 μl of the mixture containing serum-free RPMI-1640 and Matrigel™ (1:8; BD Biosciences, Bedford, MA, USA). This was allowed to solidify at 37°C overnight. After 24 h, cells that had migrated or invaded through the membrane were fixed with 95% alcohol and stained with crystal violet. The number of migrated or invaded cells was quantified by counting five independent symmetrical visual fields under an Olympus BX51 microscope at 200× magnification (Olympus Corp., Tokyo, Japan).

Cells were seeded onto six-well tissue culture dishes (4×10⁶ cells/well) and grown to 95% confluence. Each confluent monolayer was wounded linearly using a 200 μl pipette tip and washed three times with phosphate-buffered saline (PBS). Thereafter, cell morphology and movement was observed and photographed at 200× magnification [Olympus E-P5 (14–42mm II R) Olympus Corp.] at 0, 12 and 24 h.

NSCLC cells not treated with E2 were initially fixed in 1.5% agarose and incubated for 15 min at room temperature (RT). Sections (3 μm) from the paraffin block were placed on to adhesive-coated slides. In a heated antigen retrieval process (24), the slides were placed in an EDTA buffer (pH 8.0) and heated for 2 min in a steamer. The slides were incubated overnight at 4°C with monoclonal mouse anti-human anti-ERβ2 (Serotec) and polyclonal goat anti-human anti-IL-12 Rβ2 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies (25) in bovine serum albumin prior to incubating with the secondary antibodies rabbit anti-mouse immunoglobulin G (IgG) k-chain specific antibody (SAB3701212; 1:100, Sigma-Aldrich) or rabbit anti-goat IgG F(ab’)2 (SAB3700244; 1:100; Sigma-Aldrich) at RT for 20 min. Color was developed in 3,3′-diaminobenzidine (DAB) solution for 10 min followed by counterstaining with Harris hematoxylin. Cells were dehydrated, coverslipped and reviewed under an Olympus BX51 light microscope (400×; Olympus Corp.), and the mean percentage of ERβ2 and IL-12 Rβ2 positive cells was counted in 10 high power fields in each group.

Cells were homogenized in a radioimmunoprecipitation assay (RIPA) buffer containing 10% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA), and protein concentrations were then quantified using a BioRad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Primary goat anti-human polyclonal anti-IL-12Rβ2 antibodies (E-20; Santa Cruz Biotechnology Inc.) were then applied to the membranes according to the manufacturer’s instructions. The membranes were washed and treated with the appropriate horseradish peroxidase-conjugated secondary antibodies (rabbit anti-goat IgG F(ab’)2; 1:100; Sigma-Aldrich). A similar process was conducted for β-actin using the mouse monoclonal anti-actin antibody (A3854; Sigma-Aldrich). The results were visualized with chemiluminescence (West Dura; CPS160; Sigma-Aldrich).

RNA was prepared with TRIzol (Life Technologies, Rockville, MD, USA). For semiquantitative RT-qPCR, complementary (c)DNA was reverse-transcribed from total RNA with oligo(dT)16 primers and murine leukemia virus reverse transcriptase (PerkinElmer, Wellesley, MA, USA). The quantities of cDNA were adjusted by quantifying the level of actin DNA. Then, the same quantities of cDNA normalized to the actin were amplified for IL-12Rβ2, using the following primers: Forward: 5′-ATCCATGCGCCTGCTAAC-3′ and reverse: 5′-GAGTGTTTGAGAGGCCTTTTCTG-3′.

After transfection (12 h), each group of cells was treated with mock (ethanol) for 24 h. Protein (500 μg) from the cell lysates was incubated with 2 μg anti-Myc antibody or normal rabbit IgG (Santa Cruz Biotechnology Inc.) for 16 h at 4°C. To each sample, 20 μl of protein A/G-agarose beads was added (Santa Cruz Biotechnology), incubated for 1 h and washed three times with RIPA buffer. Then, the complex was resolved on 10% SDS-PAGE, transferred to the membrane and blotted with anti-Flag (1 μg/ml, Sigma-Aldrich) or anti-Myc antibody. Membranes were incubated with enhanced chemiluminescence reagent (Super Signal West Pico; Pierce, Rockford, IL, USA) and exposed to autoradiographic film (Kodak, Rochester, NY, USA).

Data are expressed as the mean ± standard deviation. The quantitative data were analyzed using Student’s t-test or one-way analysis of variance. All statistical tests were two-sided. P<0.05 was considered to indicate a statistically significant difference.

To investigate the association between ERβ2 and IL-12Rβ2, their expression and distribution was analyzed with immunocytochemical technology. ERβ2 was predominantly found in the cytoplasm and the nucleus in all three NSCLC cell lines as shown in Fig. 1A. IL-12β2 expression was largely confined to the nucleus. Compared with the normal bronchial cell line, the percentage of cells positive for ERβ2 and IL-12 Rβ2 was increased by 82.23% and 82.0%, respectively (Fig. 1B), demonstrating that ERβ2 and IL-12Rβ2 protein are overexpressed in NSCLC cell lines, which is in agreement with previous studies (5,8,16,22).

To investigate the roles and mechanism of ERβ2 further, the correlation between ERβ2 and IL-12Rβ2 was investigated. In the co-immunoprecipitation assay, certain proteins were constructed and groups were set, i.e., Flag-p38MAPK and Myc-IL-12Rβ2. Through the analyses, an interaction between p38MAPK and IL-12Rβ2, rather than a mixture, was observed, indicated due to the fact that the interaction molecular weight was slightly greater than the sum of their weights (Fig. 1C).

Western blotting and RT-qPCR analysis were performed to assess IL-12Rβ2 expression in the three NSCLC cell lines, each containing p3x-ERβ2, p3x-38MAPK, p3x-ERβ2+ SB203580, blank or empty vectors. Expression of the IL-12Rβ2 protein was increased in the p3x-ERβ2 and p3x-p38MAPK groups compared with the other groups (Fig. 2A and B). Fig. 2B shows that there was a significant increase in IL-12Rβ2 gene expression in the p3x-ERβ2 and p3x-38MAPK-containing cells compared with p3x-ERβ2+ SB203580-, blank- or empty vector-containing cells. This suggests that p38MAPK is required to enable the upregulation of IL-12Rβ2 by ERβ2.

There has been controversy over the role of ERβ2 in certain cancers, for example a number of studies have reported that E2 may promote lung adenocarcinoma development as well as insulin-like growth factor type-1 (3,4,7,8). This study demonstrated a potentially protective effect of ERβ2 in NSCLC, but showed that this effect did not persist, and indeed, was reversed, in the absence of IL-12Rβ2. An MTT assay was used to detect cell proliferation in each group. It was found that compared with the blank group, cell proliferation in the p3x-ERβ2 and pXJ40-IL-12Rβ2 groups was reduced. This effect was only apparent after 48 h, and persisted at 72 h. However, there was only minimal inhibition of proliferation in the p3x-ERβ2 and pXJ40-IL-12Rβ2 groups compared with the blank and empty vector groups at 24 h (Fig. 3B). Notably, an opposite effect was observed in the p3x-ERβ2+sh-IL-12Rβ2 group, where proliferation was observed to increase significantly compared with the blank and empty vector groups.

The transwell assay was used to measure invasiveness of the cell lines. Invasiveness was observed to be significantly reduced in the p3x-ERβ2 and pXJ40-IL-12Rβ2 groups compared with the p3x-ERβ2+sh-IL-12Rβ2, blank and empty vector groups (Fig. 3C). By contrast, the p3x-ERβ2+sh-IL-12Rβ2 12Rβ2 group showed greater invasiveness compared with the blank and empty vector groups. This suggests that ERβ2/IL-12Rβ2 signaling may be important in regulating the progression of NSCLC.