A small interfering RNA (siRNA) targeting KIRREL3 and scrambled control were purchased from GenePharma (Shanghai, China). Lipofectamine® 2000 was purchased from Invitrogen (Carlsbad, CA, USA). The overexpression lentivirus and shRNA lentivirus were purchased from GenePharma. The primary antibodies against KIRREL3 were purchased from Abcam (Cambridge, UK). The primary antibodies against phospho-STAT3 (Tyr705) and STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-Actin, anti-mouse IgG, and anti-rabbit IgG horseradish peroxidase-conjugated antibodies were purchased from ZhongshanJinqiao (Beijing, China). Curcumin was purchased from Selleck Chemicals (Houston, TX, USA). STAT3 inhibitor Stattic was purchased from Sigma (St. Louis, MO, USA).
2.2 Immunohistochemistry (IHC)
Tissues were dehydrated with xylene and an ethanol series, followed by washes with TBST. Antigen retrieval was performed with sodium citrate (0.01 M, pH = 6.0). Endogenous peroxidase was blocked by using H2O2 for 10 minutes at room temperature. After washing with TBST, the KIRREL3 primary antibody (LifeSpan BioSciences, Seattle, WA, USA, 1:100) was added and tissues were incubated overnight at 4℃. They were then incubated with secondary antibody at 37℃ for 15 minutes. They were visualized by incubating with DAB, and staining was observed with microscopy. After decoloring with l% hydrochloric acid ethanol, the tissues were dehydrated and transparentized by ethanol and xylene. The tissues were sealed by neutral resin.
2.3 Cell lines and cell culture
Immortalized human esophageal epithelial SHEE and ESCC cell lines (Eca109, KYSE150, KYSE30, KYSE450, and KYSE510) were provided by the Department of Pathophysiology, School of Basic Medicine, Zhengzhou University. Eca109 and KYSE150 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (BI, Kibbutz Beit Haemek, Israel). KYSE30, KYSE450, and KYSE510 cells were cultured in DMEM supplemented with 10% FBS. All of these cells were maintained at 37℃ in a 5% CO2 environment.
2.4 siRNA transfections
Lipofectamine® 2000 reagent was used for all siRNA transfection procedures at a final concentration of 10 µM. For immunoblotting analysis, 6.5×105 Eca109 cells and 5×105 KYSE150 cells were seeded into 6-well plates and incubated overnight. They were transfected with siRNAs for 72 hours and then collected. For apoptosis assays, Eca109 and KYSE150 cells were seeded into 6-well plates and incubated overnight. After siRNA transfections, the cells were cultured for 48 hours and collected for apoptosis analysis.
2.5 Lentivirus infections
To establish stable cell lines that overexpressed or knocked down KIRREL3, we obtained appropriate lentiviruses from GenePharma. Transfections of cells were done according to manufacturer instructions. After target cells were infected, stably transfected cells were obtained by screening with puromycin (1 µg/mL for Eca109 cells; 2 µg/mL for KYSE150 and KYSE30 cells).
2.6 Immunoblotting analysis
Cells were washed two or three times with ice-cold phosphate-buffered saline (PBS). Proteins were extracted with protein lysis buffer. Protein concentrations were measured with the BCA Protein Concentration Kit (Solarbio, Beijing, China). Samples were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was incubated with primary antibody overnight (~ 16 hours) at 4℃. The primary antibodies included KIRREL3 (Abcam, 1:1000), p-STAT3 (Tyr705) (Cell Signaling Technology, 1:1000), STAT3 (Cell Signaling Technology, 1:1000), and β-actin (Zhongshan jinqiao, 1:1000).
2.7 Cell proliferation assay
Cells (5,000 cells/well) were seeded into 96-well plates and cultured overnight. Proliferation was measured by CCK-8 assay. After being cultured for 0, 24, 48, 72, or 96 hours, 10 µL of CCK8 reagent (Beyotime, Shanghai, China) was added to each well. Following incubation for 1 hour, absorbance values at 450 nm were measured.
2.8 Cell colony formation assay
Eca109 and KYSE150 cells were seeded into 6-well plates at 300 cells/well and KYSE30 cells were seeded into 6-well plates at 400 cells/well. The cells were cultured for one to two weeks until colonies appeared. The medium was then removed and the cells were washed with PBS, followed by fixation in each well with 4% paraformaldehyde (PFA) for 30 minutes. Finally, cells were stained with 0.1% crystal violet (CV) for 30 minutes.
2.9 Transwell cell invasion and migration assays
Cell invasion ability was evaluated by Transwell cell invasion assays. An invasion assay was performed using Transwell Matrigel (Corning, Corning, NY, USA). Cells (5×105 cells/well for KYSE150 and Eca109 and 3×105 cells/well for KYSE30) were seeded into the upper chamber of the plates in RPMI-1640 or DMEM (serum-free) after siRNA transfection or lentivirus infection. RPMI 1640 or DMEM with 10% FBS was then added to the lower chamber. After 30–40 hours of incubation at 37℃ with 5% CO2, the Matrigel and cells in the upper chamber were discarded.
The medium was removed and the cells were washed with PBS, followed by fixation in each well with 4% PFA for 30 minutes. Finally, cells were stained with 0.1% CV for 30 minutes. Transwell plates without Matrigel were used for migration assays, with the rest of the procedure remaining the same.
2.10 Cell cycle assay
Cells were added to 75% ethyl alcohol that was pre-cooled overnight. After washing the cells once with PBS, 1 ml PBS was added to resuspend the cells. Then, 2.5 µL RNase (10 mg/mL) (Meilunbio, Dalian, China) was added and incubated at room temperature for 1 hour. Finally, 50 µL propidium iodide (PI) (1 mg/mL) (Meilunbio) was added and incubated at room temperature or 4℃ in the dark for 15–20 minutes. The cells were analyzed using flow cytometry.
2.11 Cell apoptosis assay
Apoptosis levels were detected 48 hours after siRNA transfections. The cells were stained with an Annexin V-FITC/PI kit (Meilunbio). Cells were digested with trypsin without EDTA and washed with PBS. The cells were resuspended with 100 µL 1× Binding buffer, then 5 µL Annexin V-FITC and 6 µL PI were added to the cell suspension, mixing well and protected from light for 20 minutes at room temperature. The proportion of early or late apoptotic cells was measured by flow cytometry.
2.12 Tumor xenograft experiments
Female BALB/c nude mice were purchased from the Beijing Weitonglihua (Beijing, China). Four-week-old female mice were maintained in specific pathogen-free conditions. The nude mice were subcutaneously injected with 5×107 Eca109 or KYSE-150 cells stably transfected with KIRREL3 or the empty vector. The mice weight and tumor growth level were measured three times a week. Tumor volume was calculated as length×width2 ×1/2.
2.13 Statistical analysis
All statistical analyses were performed using SPSS version 17.0 (SPSS, Chicago, IL, USA). The data were analyzed by one-way analysis of variance (ANOVA) or Student t test. Data are expressed as means ± standard deviation (SD). The statistically significant criterion was p < 0.05.