Oncomine analysis
We used Oncomine (https://www.Oncomine.org) [15], a publicly accessible online database of cancer gene expression profiles, to retrieve STEAP1 and STEAP2 mRNA expression in various cancers.
Kaplan-Meier (KM) plotter analysis
KM plotter (http://kmplot.com/analysis/) includes the survival data of patients with breast cancer (6234 cases), ovarian cancer (2190 cases), lung cancer (3452 cases), and gastric cancer (1440 cases) [16]. The KM plotter was used to evaluate the expression of STEAP1 and STEAP2 and their relationship with the prognosis of lung cancer patients.
GEPIA dataset analysis
Gene expression profile interactive analysis (GEPIA) (http://gepia.cancer-pku.cn/) provides fast and customizable functionality based on TCGA and GTEx data [17]. This investigates the correlation between STEAP1 and STEAP2 expression and the relationship between STEAP1 and STEAP2 and the stage of lung cancer.
GeneMANIA analysis
The interaction between STEAP1 and STEAP2 at the gene level was analyzed using GeneMANIA (http://genemania.org) [18], a network tool for identifying intra-genomic associations.
STRING analysis
STRING (https://string-db.org/) is a database used to search for the physical interactions between proteins and the functional correlations between proteins. We used STRING to analyze the protein correlations between STEAP1 and STEAP2.
Cell lines
In this study, the human lung cancer cell lines A549, H460, and H1299 and the normal lung epithelial cell line BEAS-2B were used. All chemicals were purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The A549, H460, and H1299 cell lines were grown in RPMI-1640 (Sigma Aldrich, St Louis, MO, USA) containing 10% fetal bovine serum (FBS; Clark Bioscience, Richmond, VA, USA) and 1% penicillin-streptomycin solution (Merck, Kenilworth, NJ, USA). BEAS-2B cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS and 1% penicillin-streptomycin solution and maintained in a 5% CO2 atmosphere at 37°C.
Tumor tissue samples
After obtaining informed consent from the patients, lung tissue samples were collected from the Shandong Provincial Hospital and Qilu Hospital (Shandong, China). Two pathologists examined all specimens. A total of 298 samples were obtained, including 40 normal lungs, 133 squamous cell carcinomas, and 125 adenocarcinoma samples. All lung cancer patients were diagnosed based on the tumor, lymph node, and metastasis (TNM) staging system, and no treatment was performed before tissue samples were collected. This study was approved by the Medical Ethics Committee of the Shandong University. All methods were performed in accordance with relevant standards and regulations.
Transwell migration and invasion assay
Transwell chamber invasion experiment: Matrigel was diluted with serum-free medium, and 50 µL was evenly coated on the upper chamber filter membrane of the Transwell chamber. Cells in each group were digested with trypsin and resuspended in a serum-free medium. A 200 µL cell suspension was added to the upper chamber, and 10% FBS was added to the lower chamber. After incubation at 37°C and 5% CO2 for 24 h, the cells on the upper layer of the filter membrane were removed, and the cells on the lower surface were fixed with 4% paraformaldehyde. After staining with crystal violet, the cells were dehydrated and sealed. The cells were counted in five random fields to quantify cell invasion ability.
Transwell chamber movement experiment: The filter membrane of the upper chamber of the Transwell chamber was not coated with Matrigel, and the other steps were the same as in the Transwell chamber invasion experiment described above.
Immunohistochemistry
Tissues were fixed using 4% formaldehyde, embedded in paraffin for sectioning, and removed using xylene for 5 min. The samples were then rehydrated using a graded series of alcohol dilutions. Antigen recovery was performed in citrate buffer at 125°C in a steam pressure cooker for 2 min. Streptomyces biotin protein-peroxidase staining was performed according to the manufacturer’s instructions and summarized as follows: STEAP1 antibody (1:400 dilution in phosphate-buffered saline [PBS]; cat. no. ab207914, Abcam, Cambridge, United Kingdom); STEAP2 antibody (1:100 dilution in phosphate-buffered saline [PBS]; cat. no. ab207914, Abcam, Cambridge, United Kingdom); incubation at 4°C overnight; addition of a secondary antibody (Conway Century Company) for 30 min; DAB color; hematoxylin re-dyeing; dehydration seal; and observation of tissue samples using an optical microscope. The staining results were evaluated and graded according to the ratio of staining intensity and the proportion of positive cells in tissue sections or cell climbing slices. Dyeing intensity was scored as follows: 0, no staining; 1, low strength; 2, medium strength; and 3, high strength. Positive cell ratio was divided as follows: 0% 0, 1–25% 1, 26–50% 2, 51–75% 3, 76–100% 4. The total score was represented by the sum of the staining intensity score and positive cell ratio score (0–7), with a total score ≤ 3 indicating low expression and ≥ 4 indicating high expression. All tissue sections and cell climbing results were blindly evaluated by two researchers. Differences in scoring were resolved through discussion.
Immunocytochemistry
Cultured cell lines were treated with trypsin and centrifuged to recover the cells. The cells were inoculated into 24-well plates containing cell slides and cultured in medium for 24 h at 37°C. After the cells reached 60–80% confluence, they were treated with STEAP1 and STEAP2 for immunocytochemistry, DAB staining, and hematoxylin re-staining, respectively. Staining intensity and percentage of stained cells were observed after dehydration and sealing. The rating standards were determined using immunohistochemical methods.
Real-time quantitative polymerase chain reaction
RNA was extracted from the cell lines using the TRIzol method using a commercially available kit in accordance with the manufacturer’s instructions. The concentration and purity of RNA were determined using a microplate reader. In a 20 µL reaction system using PrimeScript RT kit with gDNA eraser (Takara Bio Inc., Shiga, Japan), 2 µg of total RNA was used as a template to reverse transcribe the complementary DNA (cDNA). According to manufacturer’s instructions, a LightCycler 480 system (Applied Biosystems Inc., Waltham, MA, USA; Roche, Inc., Basel, Switzerland) was used for real-time quantitative polymerase chain reaction (RT-qPCR), which was performed in triplicate samples in 96-well plates. The qPCR mixture volume in each well was 20 µL, including 10 µL SYBR Premix EX Taq, 0.4 µL PCR forward primer, 0.4 µL PCR reverse primer, 2 µL cDNA, and 7.2 µL sterile water. Specific primers were designed and synthesized by TaKaRa Biotechnology Co., Ltd. (Japan). Primer sequences included the upstream primer for STEAP1,5'-ACAAGTTGCTAAACTGGGCATATCA-3', the downstream primer, 5'-CAGTATTGCCAATCCCACAATTC-3';STEAP2, 5′-CGCTATGGTCCATGTTGCCTA-3, downstream primer, 5′-CCAAGGCTCATTATGCCAAAG-3, an internal reference ACTB upstream primer, 5-TGGCACCCAGCACAATGAA-3, and downstream primer 5- CTAAGTCATAGTCCGCCTAGAAGCA-3′. The experiment was repeated three times.
Western blotting
Cells were collected and lysed on ice to extract protein using radioimmunoprecipitation assay buffer (i.e., “RIPA”) containing 1 mM phenylmethylsulfonyl fluoride. Protein concentration was determined using the bicinchoninic acid assay (i.e., “BCA”) method. Protein samples (30 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated protein samples were transferred to PVDF membranes and blocked with 5% bovine serum albumin. The membrane was then incubated with mouse anti-STEAP1 primary antibody (Abcam, cat. no., ab207914) and mouse anti-STEAP2 primary antibody (AbCam, cat. no., ab207914) or anti-β-actin primary antibody at a dilution of 1:2000 overnight at 4°C. The next day, the membrane was incubated with a secondary antibody at room temperature for 1 h, and the substrate was analyzed using an enhanced chemiluminescence assay (Pierce ECL western blotting substrate; Millipore, Inc., Burlington, MA, USA) and Amersham Imager 600 (GE Healthcare, Milwaukee, WI, USA) chemiluminescence models to visualize imprinting. After the membrane was washed with TBST and developed using the ECL method, the gray value was determined using ImageJ version 1.46r (National Institutes of Health, Bethesda. MD, USA) and normalized to the gray value of β-actin.
Statistical analysis
Statistical analysis was performed using SPSS (version 20.0; IBM Corporation, Armonk, NY, USA). Quantitative data were assessed using analysis of variance. Count data are expressed as percentages. The Pearson chi-squared or Fisher’s exact test was used for between-group comparisons; differences were considered statistically significant at P < 0.05.