Cell culture
Human cell lines A549, PC9, H1299, H1975, and H1650 were obtained from the American Type Culture Collection (ATCC, https://www.atcc.org/). Cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) and maintained in a 37°C incubator with a humidified atmosphere containing 5% CO2.
siRNAs and cell transfection
Cells were plated at specific densities and transfected with 10 nM siRNA oligonucleotides or non-targeting controls 24 to 48 h after plating. Transfection was performed with Lipofectamine® RNAiMax Reagent (Thermo Waltham, USA) in OptiMEM medium following the manufacturer’s instructions.
Knockdown efficiency was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with the following primers: Si-LINC00885-1: sense CCUCCCAAUUAUCUCCUCUTT, antisense AGAGGAGAUAAUUGGGAGGTT; and si-LINC00885-2: CCGGCUGGUUCAAGAUCAATT, antisense UUGAUCUUGAACCAGCCGGTT.
Cell proliferation and colony formation assays
Cells were seeded in 96-well plates at 1000 cells per well and transfected with 10 nM siRNA or non-targeting (negative) control siRNA and LINC00885 overexpression or vector 12 to 24 h after seeding. At 72 to 96 h post-transfection, the proliferation rates were measured using cell proliferation reagent (WST-1, Beyotime, Shanghai, China) following the manufacturer’s instructions. The cell viability was calculated as a percentage of the viability of the negative control siRNA–transfected cells.
For colony formation assays, cells transfected with LINC00885-targeting siRNAs or control siRNAs were seeded in a 6-well plate at a density of 250 cells/well. After 12 to 14 days of culture at 37°C, 0.1% crystal violet (Solarbio, Beijing, China) and 20% methanol were used as dye solution to fix and stain the colonies. The cells were imaged and counted.
Western blot assay
RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime) was used to prepare whole cell lysates. Protein concentration was evaluated using a BCA Protein Assay Kit (Beyotime). Proteins were separated using SDS-PAGE and then transferred to polyvinylidene difluoride membranes. After blocking membranes with skim milk, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibody for 1 h at 37°C. Protein signals were detected by P-ECL star (EpiZyme, Shanghai, China). GAPDH was used as an endogenous control.
RNA extraction and qRT-PCR
Total RNA was extracted from cultured cells using Trizol reagent (Solarbio) following the manufacturer’s instructions. Reverse transcription to cDNA was performed using a reverse transcription kit (Takara, Tokyo, Japan). qRT-PCR reactions were run in an Applied Biosystems 7500 Real-Time PC System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq II (Takara, Dalian, China). GAPDH mRNA was used as an internal control, and data were analyzed using the 2 − ΔΔCt method. Each experiment was performed in triplicate. The PCR primer sequences are as follows: LINC00885F: CCAGCAGGGCCTAGTAACAC and LINC00885R: CCTTGCTCTTGGTGAGTGGT.
Cell migration and invasion assays
Quantitative analysis of the migration and invasion capabilities of LUAD cells was performed using the Transwell chamber system. A total of 60 µL of diluted extracellular matrix gel solution was added to the upper chambers (Costar Inc., USA) and the chambers were incubated for 4 h at 37°C. For the migration assay, ECM solution was not added in the upper chamber. Cells were seeded at a density of 1×105 cells in 100 µL RPMI-1640 medium with 1% FBS; the wells were then filled with 500 µL RPMI-1640 medium containing 20% FBS. The Transwell chambers were then incubated for 24 to 36 h at 37°C with 5% CO2 to allow cell migration or invasion. When the incubation period ended, the remaining cells in the upper chamber were removed with a cotton swab. Cells that reached the bottom of the membrane were fixed with paraformaldehyde. The fixed cells were stained with crystal violet at room temperature for 30 min. The crystal violet bound to the cells was washed off with 200 µL of PBS. Cells were observed under a microscope at 4X and 10X and photographed.
Flow cytometric analysis of apoptosis and cell cycle distribution
For apoptosis analysis, cells were resuspended in moderate binding buffer containing Annexin V–fluorescein isothiocyanate (FITC) with propidium iodide (PI) (BD, NJ, USA). After incubation for 15 min, the cells were analyzed using a BD FACSAria3.
For cell cycle analysis, cells were fixed at 4°C overnight in 75% ethanol and then stained with RNase A and PI for 30 min at 37°C (Keygen, Nanjing, China). Samples were analyzed by a flow cytometer (BD Biosciences, San Jose, CA, USA) in the Guangdong Medical University flow cytometry core.
Animal models
For the subcutaneous tumor model, the armpits of mice were subcutaneously injected with transfected A549 cells (5×106 cells/mice, n = 6/group for knockdown groups). In the experiment, mice were randomly assigned to two groups: control shRNA (sh-NC) and LINC00885 shRNA (sh-LINC00885). Tumor growth was observed and measured with a vernier caliper every week. T umor volume was calculated with the formula: V = (length×width2)/2. At 4 weeks after tumor formation, mice were euthanized and subcutaneous tumors.Four-week-old female nude mice (BALB/c) were purchased from the medical experimental Animal Center of Guangdong province (Guangdong, China).
Statistical analysis
Data were analyzed using GraphPad Prism 6 and R software. Receiver operating characteristic (ROC) curve and the area under the curve (AUC) analysis were used to evaluate the tradeoff between sensitivity and specificity for the different possible cut-off points of a diagnostic test. Kaplan–Meier survival curves with log-rank test were used for survival analysis. The other datasets were evaluated by unpaired Student t-tests. A two-tailed P-value < 0.05 was considered statistically significant.
Data availability
All data generated or analyzed during this study are included in this published article.