Tissue samples
Twenty pairs of LUAD specimens and adjacent non-tumor tissues were acquired from patients who did not receive any therapy before undergoing operation in Sir Run Run Hospital of Nanjing Medical University. Tissue specimens were obtained from patients who signed informed consent. Research was performed in accordance with the Declaration of Helsinki and approval was obtained from the Ethics Committee of the Sir Run Run Hospital of Nanjing Medical University. Immediately after the operation, tissue samples were frozen in liquid nitrogen and maintained at −80 °C.
Cell culture
LUAD cell lines (A549, H1975, H2030, H1435) and a normal human bronchial epithelial cell (BEAS-2B) were provided by the American Type Culture Collection (ATCC; Gaithersburg, MD, USA). Briefly, all cells applied in this study were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 100 U/ml penicillin (Sigma-Aldrich, USA), and 100 mg/ml streptomycin (Sigma-Aldrich, USA) at 37 °C with 5% CO2.
Cell transfection
To construct pcDNA3.1/LINC00520 or forkhead box P3 (FOXP3) vector, LINC00520 (or FOXP3) were synthesized and subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) plasmid. To silence LINC00520, short harpin RNAs (shRNAs) for LINC00520 (sh-LINC00520#1/2), FOXP3 (sh-FOXP3#1/2) and their negative control (sh-NC) were purchased from GenePharma Co., Ltd (Shanghai, China). As for miR-3611, A549 and H1975 cells were transfected with miR-3611 mimics (inhibitor) or NC mimics (inhibitor) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). MiR-3611 mimics (inhibitor) and NC mimics (inhibitor) were also supplied by GenePharma (Shanghai, China). A549 and H1975 cells were transfected for 48 h. Additionally, RT-qPCR was applied to testify transfection efficiency.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from frozen tissue samples or cultured cells using Trizol reagent (Thermo Fisher Scientific) and was reverse-transcribed into complementary DNA (cDNA) using a Reverse Transcription Kit (Invitrogen). RT-qPCR analysis was conducted with SYBR Green Premix PCR Master Mix (Roche, Mannheim, Germany) by an ABI HT9600 (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was taken as the internal reference for LINC00520 and FOXP3. RNU6 (U6) was taken as the internal reference for miR-3611. The 2-△△CT method was utilized to calculate the relative quantification.
CCK-8 assay
After transfection, cell viability was measured by Cell Counting Kit-8 (CCK‐8; Dojindo, Kyushu, Japan) under the manufacturer’s guidance. A549 or H1975 cells (1 × 103) were plated into 96-well plates for 0, 1, 2, and 3 days. CCK- 8 solution was added into plates to cultivate cells for 2 h at 37 °C. Optical density at a wavelength of 450 nm was measured by a microplate reader (Thermo Fisher Scientific).
Colony formation assay
A549 or H1975 cells (1 × 103) were plated into 6-well plates at 37 °C with 5% CO2. After 2 weeks, colonies were fixed by 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min, and dyed by crystal violet (Beyotime, Nantong, China) for 5 min when colonies were visible. The plates were photographed, and the number of colonies was counted.
Flow cytometry analysis
Transfected cells were collected and resuspended with phosphate buffered saline (PBS). After being cultured at 6-well plates for 48 h, A549 or H1975 cells were fixed in 70% ethanol pre-cooled with ice for 2 h. Quantification of apoptosis was measured by flow cytometry (Thermo Fisher Scientific) after staining with Annexin V-FITC/PI (BD Biosciences, San Jose, CA, USA).
Wound healing assay
After transfection, A549 or H1975 cells seeded in 6-well plates were subjected to serum starvation for 4 h. Thereafter, the wound was stimulated by straight scratching in the cell monolayer using a sterile 200-μl pipette tip. After gently scraping the scratched monolayer cells twice with serum-free medium, the wound was healed in complete medium for 24 h. Then, after the wound was formed, photographs of the wound width at 0 and 24 h were captured using an inverted microscope, respectively. LUAD cell migration was assessed by checking the percentage of wound closure.
Western bolt
Cells were lysed with lysis buffer containing protease inhibitors (50 mM Tris-HCl pH 8; 50 mM NaCl; 0.5% NP-40). Total protein was extracted from tissues and cells using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined using a bicinchoninic acid assay. The extracted total protein (20 µg) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Solarbio, Beijing, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, Shanghai, China). After PVDF membranes was blocked with 5% skim milk at 25°C for 1 h, the primary antibodies were added for incubation overnight, including anti-E-cadherin (Abcam), anti-N-cadherin (Abcam) and anti-GAPDH (Abcam). After adding secondary antibody, proteins were visualized by an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, UK) and a Bio-Rad image analysis system (Bio-Rad, CA, USA).
DNA pull down
A DNA pull-down test kit (Gzscbio, Guangzhou, China) was utilized under manufacturer’s guidance. Biotin-labeled promoter bound with streptavidin magnetic beads were cultivated with cellular protein extracts at 4 °C overnight, and separated by SDS-PAGE, detected by RT-qPCR.
Chromatin immunoprecipitation (ChIP) assay
A chromatin immunoprecipitation (ChIP) assay was carried out with the Magna ChIP Kit (Millipore, Billerica, MA) under manufacturer’s instruction. The following antibodies were utilized to immunoprecipitated crosslinked protein-DNA complexes prior to RT-qPCR analysis using rabbit anti-FOXP3 and normal rabbit IgG.
Luciferase reporter assay
The wild-type plasmids of FOXP3 3’UTR containing the putative binding site of miR-3611 and their mutations were cloned into the pmiRGLO dual-luciferase vector (Promega, Madison, WI, USA), termed FOXP3-WT/MUT. The plasmids were co-transfected with indicated plasmids into A549 or H1975 cells with Lipofectamine 2000 (Invitrogen). The promoter sequences of LINC00520 were subcloned into pGL3 luciferase vector to construct promoter luciferase plasmids, and then co-transfected with indicated plasmids into A549 or H1975 cells. Dual-Luciferase Reporter Assay System (Promega) was utilized to confirm Luciferase activities.
Subcellular fractionation
The cytoplasmic and nuclear extracts were extracted from A549 or H1975 cells by Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). RNAs isolated from nucleus or cytoplasm were performed RT-qPCR analysis. The levels of U6 (nucleus control), GAPDH (cytoplasm control), and LINC00520 were respectively determined.
RNA immunoprecipitation (RIP) assay
RNA immunoprecipitation (RIP) experiment was conducted through the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA) under manufacturer’s instruction. Ago2 antibody was applied for RIP (Cell Signaling Technology, Beverly, MA). RT-qPCR was utilized to detect co-precipitated RNAs. IgG controls were assayed simultaneously to demonstrate that the detected signals were the result of RNAs specifically binding to Ago2.
Statistical analysis
Data of triplicate experiments were analyzed by SPSS (SPSS Inc., Chicago, IL, USA). Results acquired were denoted as means ± standard deviation (SD). Kaplan Meier and Log-rank test were performed for survival curve. Comparison between two groups was evaluated by Student's t test or comparison among three groups was assessed with analysis of variance (ANOVA) followed by Turkey’s post-hoc test. Each experiment was repeated three times in triplicate. p < 0.05 was considered as statistically differential significance.