PELP1/MNAR antibodies for western blot analysis were purchased from Abicam (Beverly, MA, USA), PELP1/MNAR antibodies for immunocytochemistry were purchased from Novus Biologicals (Littleton, CO, USA), horseradish, peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA) and GAPDH was purchased from Biosynthesis Technology (Beijing, China). ERα and ERβ antibodies were purchased from Dako (Glostrup, Denmark). 17-Estradiol (purity ≥98%; cat. no. E8875) was purchased from Sigma Chemicals (St. Louis, MO, USA).

HEC-1A and HEC-1B human endometrial carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). AN3CA and RL-952 cells were provided by the Shanghai Cell Collection, Chinese Academy of Sciences (Shanghai, China). Ishikawa human endometrial carcinoma cells were kindly provided by Professor Zehua Wang (Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China). The cells were grown in Dulbecco's modified Eagle's medium with essential amino acid solution (Gibco, Carlsbad, CA, USA) without phenol red and supplemented with 10% fetal calf serum (HyClone, South Logan, UT, USA).

To detect the expression of PELP1/MNAR protein in different endometrial carcinoma cell lines, we performed western blot analysis, and used cervical cancer HeLa cell as the control. Ishikawa, HEC-1A, HEC-1B, AN3CA, RL-952 and HeLa cells were lysed using the KeyGen Total Protein Extraction kit (KeyGen, Nanjing, China) according to the manufacturer's instructions. The lysates were centrifuged at 4°C 14,000 rpm for 20 min. The supernatants were collected and the concentrations of the supernatants were measured with a BCA protein assay kit (Pierce, Rockford, IL, USA). Protein (25–30 μg) was applied per lane and separated by 8–12% SDS polyacrylamide gel electrophoresis, then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with blocking buffer for 60 min at room temperature. The blocking buffer consisted of 5% non-fat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). After washing the membranes with TBS-T, they were immunoblotted overnight at 4°C and were incubated with rabbit anti-human PELP1/MNAR (1:300), at 4°C overnight. The membranes were washed with TBS-T 3 times, and subjected to HRP-conjugated secondary antibody for 60 min at room temperature. Protein-antibody complexes were visualized with an ECL western blot detection system (Pierce). Western blot films were digitized, net band intensities were quantified using the Quantity One Image System and GAPDH expression levels were used to further normalize the loading amount. Each experiment was repeated 3 times to assess the consistency of the results.

For immunocytochemical staining, the Ishikawa, HEC-1A and AN3CA cells were plated on coverslips and cultured for 48 h. The coverslips were fixed in 4% paraformaldehyde for 30 min at room temperature, washed in PBS, and permeabilized for 10 min with 0.25% Triton X-100 in PBS. The cell coverslips were then incubated with 0.3% H₂O₂ in PBS for 15 min. After washing in PBS, the samples were then incubated with rabbit polyclonal anti-human PELP1 antibody (1:500), mouse anti-human ERα antibody (1:60), or rabbit anti ERβ (1:60) for 1 h in a humid chamber. After washing with PBS, the sections and coverslips were overlaid with Dako REAL EnVision HRP rabbit/mouse antibody at 37°C for 30 min. The sections and coverslips were then counterstained with hematoxylin. Positive reactions were detected by incubating the slides with 3,3′-diaminobenzidine tetrahydrochloride for 1 min. The immunocytochemical results were evaluated by a pathologist.

DNA template oligonucleotides corresponding to the PELP1/MNAR gene (GenBank NM_014389) were designed (28) and synthesized. The targeted sequences were: PELP1/MNAR-shRNA, 5′-GGACCAAGGTGTATGCG ATAT-3′; the sequence was inserted into the AgeI and EcoRI enzyme sites of the pGCSIL-GFP vector. The negative control (NC) sequences were 5′-TTCTCCGAACGTGTCACGT-3′. The lentiviral particles were produced using a Lentiviral Vector Expression System (Auragene, Changsha, China). Briefly, the vectors with helper plasmids were transfected into 293T cells using the calcium phosphate transfection method. The supernatant containing lentiviral particles was collected and concentrated by ultracentrifugation. The condensed lentiviral particle solution was tittered on 293T cells with the final titer ~5×10⁸ TU/ml.

For transfection, 2×10⁵ cells/well were seeded in a 6-well plate for 24 h. Cells were then transfected with lentivirus-mediated shRNA targeting PELP1/MNAR or negative control lentivirus vector. The multiplicity of infection (MOI) was 50. Cells transfected with lentivirus-mediated shRNA targeting PELP1/MNAR, negative control and with no transfection were named Ishikawa-KD, Ishikawa-NC and Ishikawa-CON cells, respectively. The transfection efficiency was evaluated by fluorescence microscopy and flow cytometry.

Total RNA was isolated from the cells using TRIzol (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized using a PrimeScript™ RT reagent kit (Perfect Real-Time; Takara, Beijing, China). The reverse transcription (RT) reaction mixture for first-strand cDNA synthesis included 5X PrimeScript™ Buffer 2 μl, PrimeScript™ RT Enzyme mix I 0.5 μl, Oligo(dT) primer 25 pmol, random 6 mers 50 pmol, total RNA 500 ng and RNase free dH₂O in a final volume of 10 μl. The samples were placed on ice before reverse transcription. Then tubes were incubated for 15 min at 37°C, 5 sec at 85°C and then chilled to 4°C.

PCR was carried out in a 20 μl final volume containing the following: 2 μl cDNA diluted in RNase-free water; 10 μl SYBR premix Ex Taq; the antisense and sense primers 0.5 μl separately; ROX Reference dye II (50X) 0.4 μl and dH₂O in order to reach a 20 μl final volume. After an initial denaturation step at 95°C for 30 sec, temperature cycling was initiated. Each cycle consisted of denaturation at 95°C for 3 sec and elongation at 60°C for 30 sec. A total of 40 cycles was performed. The fluorescent signal was acquired at the end of the elongation step. Real-time PCR was performed in a ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with specific real-time PCR primers for each gene (Table I). Results were normalized to the actin transcript levels and the difference in fold-expression was calculated using the ΔΔCT method. The results were representative of 3 independent experiments. Data are shown as the means ± SD. The primers for PELP1/MNAR, β-actin, and MMPs are shown in Table I.

The cell viability and proliferation were measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA). Seven days after the transfection the 3 types of Ishikawa cells were seeded into 5 96-well culture plates at 3.0×10³/well, with each group consisting of 5 parallel wells. After 24 h, the cells were treated with or without E2 (10⁻⁹ mol/l). For the following 5 days, 20 μl MTT (5 mg/ml) were added to each well and the cells were incubated at 37°C for an additional 4 h. The reaction was terminated by lysing the cells with 150 μl DMSO for 10 min. Absorbance was measured at 490 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Cell growth curves were drawn by the mean optical density (OD) values.

Five days after the transfection, the 3 types of Ishikawa cells were plated in 10-cm culture dishes at a density of 3×10³/dish, respectively. After 24 h, the cells were treated with or without E2 (10⁻⁹ mol/l). The cells were fed every 2 days, and the foci of the transformed cells were counted after 14 days. The cells were stained with crystal violet and visible colonies were manually counted. The data are representative of 3 independent experiments performed in triplicate.

Cell migration and invasion assays were all performed in a 24-well, Transwell chamber with a 8.0-μm pore membrane (Corning, Corning, NY, USA). For the cell migration assay, 3 types of Ishikawa cells (5×10⁴) were seeded in the upper chamber with serum-free medium, and 0.75 ml of complete growth medium containing 10% fetal bovine serum with or without E2 (10⁻⁹ mol/l) was added to each well in the lower chamber (21). For the invasion assays, the upper chambers were coated with 20% Matrigel (100 μl/well) (BD Biosciences, San Diego, CA, USA). The cells were then seeded in the chamber at 1×10⁵/well and treated as described above. After 24 h of incubation at 37°C in a 5% CO₂ atmosphere, cells on the upper side of the chamber were removed and cells invaded to the lower side were fixed, stained and counted in 10 random fields at magnification, ×100.

All assays were conducted 3 times and found to be reproducible. Data are expressed as the means ± SD. Comparisons among the groups were performed with the one-way analysis of variance (ANOVA) test with Bonferroni's correction for multiple comparisons. All statistical analyses were performed by using SPSS 13.0. A P-value <0.05 was considered to indicate a statistically significant difference.

To determine whether PELP1/MNAR plays a role in endometrial cancer, we first measured the PELP1/MNAR expression in 5 commonly used endometrial cancer cell lines (Ishikawa, HEC-1A, AN3CA, HEC-1B and RL-952) and cervical cancer HeLa cells using western blot analysis. The results revealed that PELP1/MNAR was widely expressed in all the cell lines (Fig. 1). We then used immunocytochemistry to detect PELP1/MNAR as well as ERα and ERβ expression and location in the Ishikawa, HEC-1A and AN3CA endometrial cancer cells, and found that the expression of ERα and ERβ varied among the cells. Ishikawa cells were ERα-positive, HEC-1A cells were ERβ-positive, whereas ERα and ERβ expression was low/undetectable in AN3CA cells (Fig. 2). However, all the cell lines were stained with PELP1/MNAR and the protein was mostly located in the nucleus, which implies a potentially important role for PELP1/MNAR in endometrial cancer. Therefore, we further investigated the effects of PELP1/MNAR suppression on the Ishikawa endometrial cancer cell line and the signaling pathway involved.

To examine the function of endogenous PELP1/MNAR in endometrial cancer cells, we selectively knocked down the endogenous PELP1/MNAR expression using shRNA methodology. The fluorescent microphoto indicated that transfection was successful (Fig. 3). Additionally, flow cytometry analysis showed that the transfection efficiency was above 80%. After 5 or 7 days of tranfection, the silencing effect was examined by real-time PCR and western blot analysis in the mock and stably transfected Ishikawa cells. As shown in Fig. 4, compared with the mock-transfected Ishikawa cells, the levels of PELP1/MNAR mRNA and protein in Ishikawa-KD cells were significantly reduced by 86 and 65%, respectively (P<0.05); however, there was no significant difference between the Ishikawa-NC and Ishikawa-CON cells. These results confirmed the efficacy of lentivirus-mediated PELP1/MNAR-specific shRNA in downregulating endogenous PELP1/MNAR expression.

To further analyze the effect of PELP1/MNAR downregulation on the proliferation of Ishikawa cells, we measured the proliferation rate of Ishikawa, Ishikawa-NC and Ishikawa-KD cells with or without 17β-E2 by MTT. The results showed that the proliferation of the Ishikawa-KD cells decreased in a time-dependent manner compared with the parental cells and that the effect of PELP1/MNAR downregulation on cell proliferation was more pronounced when 17β-E2 was added (Fig. 5). We then analyzed the anchorage-dependent growth using a colony-forming assay. Similarly, Ishikawa-KD cells showed fewer colonies than Ishikawa-CON and Ishikawa-NC cells, with or without E2 (10-9 M) treatment; however, there was no difference between the Ishikawa-NC and Ishikawa-CON cells (Fig. 6).

To further examine whether the downregulation of PELP1/MNAR in Ishikawa cells reduces their migratory potential, we examined Ishikawa, Ishikawa-NC, Ishikawa-KD cells using Boyden chamber assay. Compared to the control cells, the knockdown of PELP1/MNAR in Ishikawa cells resulted in significantly reduced cell motility (Fig. 6). Moreover, the accelerated cell migratory effect of E2 was blocked in the Ishikawa-KD cells. The downregulation of PELP1/MNAR in the Ishikawa cells also significantly reduced the invasive potential, as shown by Boyden chamber assay (Fig. 7) and more importantly, abolished the E2-enhanced invasion (P<0.05). These results suggest that PELP1/MNAR plays an important role in cell invasion. To further understand the mechanism involved, we examined the expression of matrix metalloproteinase (MMP)-2 and MMP-9 to investigate whether PELP1/MNAR downregulation altered the expression of MMPs. MMPs promote cancer progression by enhancing the growth, migration, invasion and metastasis of endometrial cancer cells. Total RNA isolated from Ishikawa, Ishikawa-NC and Ishikawa-KD cells was used for real-time PCR and the results suggested that PELP1/MNAR downregulation substantially reduced the expression of MMP-2 and MMP-9 compared to their expression in the control cells (Fig. 8).