Ethics statement
The study was approved by the Human Investigation Ethics Committee of Shanghai First People’s Hospital Affiliated to Shanghai Jiao Tong University. The samples of endometrial carcinoma and normal endometrial tissues were collected after written informed consent from the patients. Animal research was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Department of Laboratory Animal Science at Shanghai Jiao Tong University School of Medicine. All efforts were made to minimize suffering.
Tissue specimens
Tissue samples for immunohistochemistry and real-time quantity PCR (RT-qPCR) were obtained at Shanghai First People’s Hospital Affiliated to Shanghai Jiao Tong University from 2011 to 2013. The stages and histological grades of these tumors were established according to the criteria of the Federation International of Gynecology and Obstetrics (FIGO) surgical staging system (2009) [19]. None of the patients underwent hormone therapy, radiotherapy, or chemotherapy prior to surgery.
Cell culture
Human endometrial cancer cell lines including Ishikawa and HEC-1B were obtained from the Chinese Academy of Sciences Committee Type Culture Collection (Shanghai, China, Additional file 3). According to the provider’s instructions, cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO2 in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Life Technologies, Auckland, New Zealand) supplemented with 10 % fetal bovine serum (Gibco, Carlsbad, CA, USA).
Ishikawa cell line is a human endometrial adenocarcinoma cell line which contains estrogen and progesterone receptors [20]. HEC-1B cell line is a human endometrial adenocarcinoma cell line which has a low baseline level of estrogen and progesterone receptors [21].
Immunohistochemistry
Tissue immunohistochemistry was performed by the 3,3'-diaminobenzidine (DAB) method with a heat-induced antigen retrieval step. Briefly, slides were incubated with rabbit polyclonal anti-Piwil1 (1:100, ab105393, Abcam), rabbit monoclonal anti-E-cadherin (1:400, #3195, CST), rabbit monoclonal anti- Vimentin (1:100, #5741, CST), rabbit monoclonal anti-CD44 (1:100, ab51037, Abcam), rabbit monoclonal anti-ALDH1 (1:100, ab52492, Abcam), rabbit monoclonal anti-ki67 (1:100, ab16667, Abcam) and rabbit monoclonal anti-PCNA (1:100, ab92552, Abcam) overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse secondary antibody (Boster) at room temperature for 30 min followed by chromagen detection with DAB (Boster) and hematoxylin (Boster) counterstaining. Isotype control antibodies was used as negative control.
Two independent pathologists, who were blinded to the clinical and pathological data, evaluated the specimens. Sections were evaluated according to semi quantitative immunoreactivity scores. We separately scored for the percentage of positive staining (0 = negative, 1 = 25 %, 2 = 25–50 %, 3 = 50–75 % and 4 = 75 %) and the staining intensity (0 = none, 1 = weak, 2 = moderate, and 3 = strong). For each specimen, the summation of the two above gave the final score.
Total RNA extraction and real-time RT-PCR
Total RNA was extracted from tissues and cell lines using Trizol (Invitrogen) and cDNA was prepared using the reverse transcriptase kit (TaKaRa) according to the manufacturer’s instructions. The cDNA was analyzed by real-time PCR using SYBR Premix Ex Taq (TaKaRa) in an Eppendorf Mastercycler realplex. A housekeeping gene, GAPDH, was used as an internal control. Data was calculated using the 2-△△Ct formula. Primers sequences are shown in Additional file 1: Table S1.
Western blotting
Cells were lysed in lysis buffer (Beyotime) for 30 min at 4 °C. Total proteins were fractionated by SDS–PAGE and transferred onto PVDF membranes (Millipore). The membranes were then incubated with primary antibodies against Piwil1 (1:1000, ab105393, Abcam), E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, #13116, CST), Vimentin (1:1000, #5741, CST), CD44 (1:5000, ab51037, Abcam), CD133 (1:1000, 18470-1-AP, ProteinTech) and ALDH1 (1:1000, ab52492, Abcam) at 4 °C overnight, followed by incubation with peroxidase-linked secondary antibody (1:10000, 112-005-003, Jackson ImmunoResearch). The probed proteins were detected by enhanced chemiluminescent reagents (Thermo). GAPDH (1:2000, #5174, CST) was used as an internal control.
Immunofluorescence
Cells were cultured on glass coverslips for 24 h and then fixed in 4 % paraformaldehyde. They were permeabilized with 0.1 % Triton X-100. After blocking in 5 % bovine serum albumin for 1 h at room temperature, cells were incubated with primary antibodies as follows: Piwil1 (1:50, ab105393, Abcam), E-cadherin (1:200, #3195, CST), Vimentin (1:100, #5741, CST), CD44 (1:100, ab51037, Abcam), CD133 (1:100, 18470-1-AP, ProteinTech) and ALDH1 (1:1000, ab52492, Abcam) overnight at 4 °C. Next, cells were incubated with Alexa Fluor 647 or rhodamine (TRITC)-conjugated secondary antibodies (1:200, Jackson ImmunoResearch) for 1 h. Nuclei were visualized by counterstaining with 496-diamidino-2-phenylindole (DAPI). Samples were analyzed using a Leica TCS SP8 confocal microscope (Leica Microsystems). Isotype control antibodies was used as negative control.
Stable transfection
HEC-1B cells were transfected with Piwil1 expression plasmids (exPiwil1, Genepharma, Shanghai, China) or control plasmids (pEGFP-N1, empty vector, EV, Genepharma) by LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s protocol. Stable overexpression clones (HEC-1BexPiwil1 and HEC-1BEV cells) were selected in the presence of 1 mg/ml G418 (Gibco) and then propagated in the presence of 0.5 mg/ml G418.
Ishikawa cells were transfected with shRNA against Piwil1 (shPiwil1, Genepharma) (sense: 5’-AGTCAGCAACCTGGTTATA-3’; antisense: 5’- TATAACCAGGTTGCTGACTGG -3’) or shRNA against nontarget (NT, Genepharma) by Lipofectamine™ 2000. Stable knockdown clones (IshikawaEV and IshikawashPiwil1 cells) were selected in the presence of 0.5 μg/ml puromycin (Sigma; St. Louis, MO, USA) and maintained with 0.3 μg/ml. Transfection efficiency was confirmed by RT-qPCR and western blot.
MTT and colony-formation assays
Cells and transfected cells (3 × 103 cells/well) were plated in 96-well plates. Then, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 5 mg/ml; Sigma) was added to each well and then incubated at 37 °C for 4 h. Absorbance values were then measured at 490 nm using a microplate reader (Bio-Red). For colony formation assay, 200 cells/well were seeded into 6-well plates. When clearly identifiable cell clones had formed, the colonies were fixed with methanol and stained with 0.5 % crystal violet. All experiments were repeated at least three times.
Cell migration and invasion assays
Cell lines were suspended in serum-free medium and plated at a density of 1 × 105 cells/well (for the migration assay) or 2 × 105 cells/well (for the invasion assay) in 6.5 mm transwell chambers equipped with 8.0 μm pore-size polycarbonate membranes without or with matrigel coating (BD Biosciences). Complete medium (600 μl) was added to the lower chamber. After incubation for 24 h (migration assay) or 48 h (invasion assay), cells were fixed in 4 % paraformaldehyde and stained with crystal violet. Then cells that migrated to the basal side of the membrane were counted at 200× magnification. The migration and invasion assays were repeated at least three times.
Spheroid formation
Cells (5 × 103) were plated on non-adherent 6-well culture plates (coated with a 10 % polyHEMA (Sigma) for 4 h and dried for 3 days at 37 °C). After plating, cells were incubated in a serum-free medium consisting of DMEM/F12 supplemented with 20 ng/ml of EGF, 10 ng/ml of bFGF, and 2 % B27 (all from Sigma and Gibco). The number of spheroids per well was counted after 5 days under light microscopy at a 200-fold magnification. The experiments were repeated at least three times.
Nude mouse tumor xenograft assay
Athymic female nude mice (BALB/c, 4 to 6 week old, n = 5 per group) were obtained from Shanghai Life Science Institute (Slac Laboratory Animal Co., Ltd, Shanghai, China). IshikawashPiwil1 or IshikawaNT cells were injected subcutaneously into the flank of each mouse at a density of 1 × 107 cells to establish a mouse model bearing endometrial cancer. The growth of tumors was monitored throughout the experiment and tumor size was measured with calipers every 4 days and the tumor volume was calculated as (Rmax) × (R2 min)/2. Four weeks after injection, mice were euthanized, tumors were removed carefully, and the weight and volume of tumors were measured.
Statistical analysis
All data analyses were performed using the software package SPSS v. 18 (SPSS Inc., Chicago, IL, USA). Values were expressed as mean ± the standard deviation and analyzed with the Student’s t-test or Mann–Whitney U test. Significant differences were indicated for P values < 0.05.