2.1 Specimen Collection
217 specimens were randomly obtained either by biopsy or complete resection from patients with NSCLC at the First Affiliated Hospital of China Medical University. According to the 2015 World Health Organization (WHO) lung cancer histopathological diagnostic criteria, 123 cases were adenocarcinoma and 94 were squamous cell carcinoma (SCC). According to the eighty edition of the American Joint Committee on Cancer TNM staging criteria published by the International Association for the Study of Lung Cancer in 2017, 135 cases were stage I-II and 82 cases were stage III-IV. None of them was treated with radiotherapy, chemotherapy, or TKIs before definitive pathological diagnoses were determined.
The present study was conducted with the approval of the China Medical University Local Institutional Review Board (2017025). Informed consent was obtained from all the patients for analysis of their specimens.
2.2 Immunohistochemistry Staining
Immunohistochemistry assays were performed as described previously [10]. Briefly, tissue sections were incubated with normal goat serum to block nonspecific antibody binding and then with mouse monoclonal anti-SIPA1L3. (1:100; Cell Signaling Technology (CST) Inc., Danvers, Massachusetts). Positive SIPA1L3 expression was determined by the intensity of cytoplasm staining of tumor cells and the proportion of SIPA1L3-positive cells in the tissue sections. The immunostaining assessment scale as follows: negative (no staining), weak (light yellow staining, no clear granular or yellow staining, and clear granular staining area <10%), moderate (yellow staining and clear granular staining area ≥10% or brown staining and clear granular staining area <10%), or strong (brown staining and clear granular staining area ≥10%). Negative to weak were considered to be normal SIPA1L3 expression and moderate to strong were considered to be SIPA1L3 overexpression. Two investigators blinded to the clinical data independently and randomly examined all tumor slides.
2.3. Western blot analysis
Western blot assays were performed as described previously [11]. Protein concentration was determined with a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). Equal amounts of lysate were separated by SDS-PAGE and western blot. The primary antibodies against YAP (#14074, 1:500), MST1 (#14946, 1:500), phospho-MST1 (#49332, 1:500), LATS1 (#3477, 1:500), phospho-LATS1 (#9157, 1:500), GAPDH (#5174, 1:10000), and AMOT (#43130, 1:500) were purchased from CST Inc (Danvers, MA, USA). Primary antibodies against LaminB1 (#ab16048, 1:1000/IB) were purchased from Abcam. Cyclin E (sc-377100, 1:200), Pals (sc-365411, 1:200), E-cadherin (sc-8426, 1:200), N-cadherin (sc-8424, 1:200), ZO-1(sc-33725, 1:200), Vimentin (sc-6260, 1:200), Snail (sc-166476, 1:200), a-SMA (sc-53142, 1:200), and CTGF (sc-34772, 1:200) were from Santa Cruz Biotechnology (Santa Cruz, California). SIPA1L3 (HPA045480, 1:500) was from Sigma (Merck Life Science (Shanghai) Co., Ltd. Shanghai, China). Secondary antibodies were purchased from CST Inc. Expression was quantified using densitometry and ImageJ software, the protein bands were visualized with ECL western blotting substrate (Pierce, Rockford, IL, USA) and the BioImaging System (UVP Inc.,Upland, CA, USA), which were normalized to GAPDH.
2.4. Cell lines and transfection
The human bronchial epithelial cells (HBEs), a human adenocarcinoma cell line (A549, LTEP-a-2, H1299), and an SCC cell line (LK-2, SK-MES-1) were purchased from ATCC (Manassas, VA, USA). The SIPA1L3-siRNA and the negative controls were purchased from Genechem Biological Technology Co. Ltd. (Shanghai, China). The cells were transfected with plasmids using Attractene transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
2.5. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cells were inoculated into 96-well plates and 5 mg mL-l MTT (Sigma-Aldrich Corp., St. Louis, MO, USA) was added to each well. Then 200 μL dimethyl sulfoxide (DMSO) was added to each well and the plates were shaken for 10 min to dissolve the formazan crystals. OD490 was measured in amicroplate reader (Model 550; Bio-Rad Laboratories, Hercules, CA, USA).
2.6. Matrigel Invasion Assay
The cell invasion assay was performed in a 24-well Transwell chamber with an 8-μm pore size (Costar, Cambridge, MA). Inserts were coated with 20 μL Matrigel (1:3 dilution; BD Bioscience). Forty-eight hours after transfection, 3×105 cells in 100 μL of serum-free medium were transferred to the upper Matrigel chamber and incubated for 16 hours, whereas 600 μL of culture medium supplemented with 10% fetal bovine serum was placed in the lower chamber. After 18 hours of incubation, the noninvaded cells were removed from the upper membrane surface with a cotton swab, and the cells that passed through the filter were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of invaded cells was counted in 10 randomly selected high-power fields under a microscope. This experiment was performed in triplicates.
2.7. Colony Formation Assay
Cells were plated into 6-cm cell culture dishes (1000 cells per dish) and incubated for 14 days. The plates were washed with phosphate buffered saline, fixed with 4% paraformaldehyde, and stained with Giemsa. Colonies containing >50 cells were counted under a microscope. Images were made and the number of colonies was established manually.
2.8. Statistical analysis
SPSS v. 22.0 (IBM Corp., Armonk, NY, USA) was used for all analyses. Each cell line experiment was performed in triplicate. The western blot gray value was detected with Image LabTM (Bio-Rad Laboratories, Hercules, CA, USA) and compared by Student’s t-test. P< 0.05 indicated statistical significance.