Patients and tissue samples
A total of 42 patients from Huaihe Hospital of Henan University (Kaifeng, China) participated in the study and provided written informed consent. None of the patients received any adjuvant therapies before the surgery. NSCLC tissues and adjacent non-cancerous tissues were collected from these patients and immediately frozen in liquid nitrogen and stored at -80°C. This study was approved by the Ethics Committee of Henan University.
Cell lines and cell culture
Human NSCLC cell lines (A549 and H1299) and the normal bronchial epithelial cell line BEAS-2B were obtained from the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin, followed by incubation in a humidified atmosphere with 5% CO2 at 37°C.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription from RNA to cDNA was performed using PrimeScript RT reagent (Takara, Japan). RT-qPCR was carried out using SYBR Green Mixture (Takara) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following primers were used: LINC00958, 5’‑CCATTGAAGATACCACGCTGC-3’ (forward) and 5’-GGTTGTTGCCCAGGGTAGTG-3’ (reverse); miR-204-3p, 5’-AGCTGTACAAGTAAGCCTGATCATGTACCCATAGG-3’ (forward) and 5’-GGGAGAGGGGCTTAGCTTATGGGACAGTTATGGGC-3’ (reverse); KIF2A, 5’-GCCTTTGATGACTCAGCTCC-3’ (forward) and 5’-TTCCTGAAAAGTCACCACCC-3’ (reverse); GAPDH, 5’-TGCACCACCAACTGCTTAGC-3’ (forward) and 5’-GGCATGGACTGTGGTCATGAG-3’ (reverse); U6, 5’-CTCGCTTCGGCAGCACA-3’ (forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse). The expression levels of target genes were normalized to internal control and calculated using the 2−ΔΔCt method.
Western blot analysis
Total protein was extracted by lysis buffer (Sigma, St. Louis, MO, USA) supplemented with PMSF (Biotool, Houston, TX, USA). Then proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After incubation in skim milk at 37°C, the membranes were incubated overnight at 4°C with primary antibodies against KIF2A and GAPDH, followed by incubation with corresponding secondary antibodies. Protein bands were detected by an ECL kit (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed by the ImageJ software.
The shRNA targeting LINC00958, miR-204-3p mimics/inhibitors and corresponding negative controls were purchased from GeneCopoeia (Rockville, MD, USA). The pcDNA3.1-KIF2A and empty vectors were from RiboBio (Guangzhou, China). Transfection of shRNAs, miRNAs or plasmids was conducted using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Cell proliferation assay
Cell proliferation was assessed using the CCK-8 assay. In brief, 2×103 cells were plated into a 96-well plate. After incubation for different time, CCK-8 solution (Dojindo, Tokyo, Japan) was added to each well and cells were further incubated for 4 h. The absorbance at 450 nm was measured using a microplate reader.
Cell migration assay
Cell migration was evaluated using transwell chambers (Corning, New York, USA). 5×104 cells in serum-free medium were added to the upper chamber and 10% FBS was added to the lower chamber. Subsequently, cells were incubated for 48 h and those migrating to the lower surface of the insert were fixed and stained with 0.1% crystal violet. The number of migrating cells from five random fields was counted under a microscope.
In vivo xenograft tumor assay
Female BALB/c mice aged 5 weeks old were obtained from Shanghai Laboratory Animal Center (Shanghai, China) and kept under specific pathogen-free conditions. All animals were handled with approval of the Animal Care and Use Committee of Henan University. A549 cells transfected with sh-LINC00958 or sh-NC were administered into the left flank of the nude mice via subcutaneous injection. Tumor volume was measured every week and calculated using the following formula: volume (mm3) = length x width2/2. Five weeks later, mice were sacrificed, tumors were weighed, and tumor tissues were dissected for further experimental analysis.
Luciferase reporter assay
Luciferase reporter vectors were constructed using pGL3 vectors (Promega, Madison, WI, USA). 1×104 cells were seeded onto a 96-well plate, followed by co-transfection with pGL3-LINC00958 (WT), pGL3-LINC00958 (MUT), pGL3-KIF2A 3’-UTR (WT), pGL3-KIF2A 3’-UTR (MUT) and miR-204-3p mimics or miR-NC using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was detected by the Dual-Luciferase Reporter Assay System (Promega).
Data were presented as means ± standard deviation (SD). Student’s t-test or one-way ANOVA was used to compare the significant difference of different groups. Statistical analysis was performed using GraphPad Prism and SPSS 20.0 software and p<0.05 was considered statistically significant.