The human H520 and HCC95 cell lines were purchased from the American Type Culture Collection. MiR-214-3p and Negative Control (NC) inhibitors and mimics were from Ribobio Technology.
Human Tissue Specimens
For research use, the LSCC samples were collected from the Department of Oncology, Shanghai Chest Hospital. Written consent forms were obtained from every LSCC patient. The Institute Research Ethics Committee of Shanghai Chest Hospital approved all protocols.
YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879)，and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scientific) and captured using the Odyssey Infrared Imaging Analysis Software.
Through the Trizol Reagent (Life Technologies, CA, USA), the total RNA was collected. According to the manufacturer’s protocol, the q-PCR was conducted using the SYBR (Synergy Brands Synergy Brands) Premix Ex Taq Kit (TaKaRa). Briefly, the cDNA (complementary DNA) was used as the template for qPCR detection using TaqMan Technology on an Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, CA). Then we detected the expression of YAP1, SOX2 (SRY-related HMG-box 2), CD133, OCT4 （Octamer-binding transcription factor-4） and NANOG genes by using commercially available primer and probe sequences (Applied Biosystems).
The miRNA mimics were synthesized through the Ribobio Technology. Both the H520 and HCC95 cells were grown in 6-well plates and transfected with Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific) in Opti-MEM (Invitrogen; Thermo Fisher Scientific). After 48 hours, cells were harvested to the following experiments.
Luciferase Activity assay
The luciferase assay was performed using the dual-Luciferase® reporter assay kit (Promega, Madison, WI). Briefly, the 293T cells were grown in 24-well plates and co-transfected with miR-NC or miR-214-3p mimics, pmirglo-H-YAP1-3’ Untranslated Region (UTR) wide type (WT) or pmirglo-H-YAP1-3’UTR-mutation type (Mut) or pGL3-YAP1 promoter, and renilla luciferase vector with Lipofectamine 2000 (Invitrogen). After 36 hours, the firefly luciferase activity normalized to the renilla luciferase.
Using the Cell Counting Kit-8 (CCK8) (Dojindo, Kumamoto, Japan), cell proliferation and viability were measured. After transfection, cells were grown into 96-well plates (1000 cells/well, N=4). The optical density value (OD value) at 450nm of the cells at the specified time was recorded.
All 500 cells with different treatments were grown into 6-well plates. The cells were cultured for at least ten days until the colonies became visible and noticeable. The cells were washed with phosphate buffer saline (PBS), fixed with 4% polyformaldehyde (Sigma-Aldrich, St. Louis, MO), and then stained with Giemsa (Solarbio, Beijing, China). Image J recorded photos of the colonies.
Sphere formation assay
Briefly, adherent H520 and HCC95 cells with different treatments were seeded into the 6-well ultra-low attachment plates at a density of 3000/well. After 14 days, spheres (>75 μm diameter) were tallied and captured. Every three days, the culture medium was refreshed, and oncosphere numbers were counted using the Leica digital camera. The serum-free DMEM-F12 medium (Life Technologies, Grand Island, NY) involves the B-27 Supplement (Life Technologies, Grand Island, NY), 20 ng/ml basic fibroblast growth factor (bFGF, Gibco), and 20 ng/ml Epidermal Growth Factor (EGF, Gibco).
All paraffin-sections of mouse lung tumors were acquired from a Leica Cryostat. The tumor sections were incubated with one of the following polyclonal antibodies: rabbit YAP1 antibody, rabbit SOX2 antibody, and rabbit CD133 antibody (all at 1:100 dilution, CST, Danvers, MA). After washing with the PBS for at least three times, the sections were incubated with the Alexa-488-conjugated secondary anti-body (1:200 dilution, Life Technologies) for 1 hour at room temperature. Furthermore, the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:500 dilution, Beyotime Institute of Biotechnology, China). All images were taken using a confocal laser-scanning microscope (Leica TCS SP5 II, Germany).
Tumor Xenograft Model
Lung squamous cells transfected with miR-NC mimics or miR-214-3p mimics were triple-washed in the PBS and then suspended in matrigel. Cells were subcutaneously injected into the armpit of each mouse. Every two days, each tumor diameter was measured. Tumor volume was calculated by the equation: tumor volume (mm3)= 1/2×length (mm)×width mm2. After nearly four weeks, tumors were all collected for subsequent analysis. All male BALB/C nude mice (SLAC, Shanghai) were fed at the specific pathogen free (SPF) Animal Center of Shanghai Chest Hospital. All experiments were performed according to the guidelines.
Orthotropic Lung Tumor Model
Cells were suspended in matrigel and orthotropically injected into the left lateral lungs of the mice, as described with minor modifications.
GraphPad Prism software was employed for statistical analysis. All results were presented as means ± Standard error of the mean (SEM) where indicated formats least three independent replicate experiments. Statistical differences were investigated using the Student’s t-test, χ2 test, and repeated measures analysis of variance. The Kaplan–Meier method was performed to assess overall survival (OS), and the log-rank test was performed to analyze the curves. Finally, the Cox model was utilized to identify independent prognostic factors. The statistical significance was set at P < 0.05.