Patients and tissues
A total of 83 paired tumor samples and corresponding normal lung tissues were collected from NSCLC patients who received surgery at our hospital. Clinicopathological information of the patients is summarized in Supplementary Table S1. None was given any anticancer treatment before surgery. Tissue specimens were immediately frozen in liquid nitrogen and stored at -80°C.
NSCLC cell lines A549, H1299, H358, and PC9 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37°C with 5% CO2 atmosphere. BEAS-2B cells were cultured in growth factor-supplemented medium (BEGM; Lonza, Walkersville, MD, USA). No mycoplasma infection was detected in the cell lines used in this study.
Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using the Superscript III Reverse Transcriptase Kit (Invitrogen). qRT-PCR analysis of ZNF674-AS1 and p21 was performed using the following primers: ZNF674-AS1 forward, 5′-GCAGTGAATTACTGCTCATTC-3′ and reverse, 5′-GCCACAGATCAGGTGCTTCT-3′; p21 forward, 5′-GCCCAGTGGACAGCGAGCAG-3′ and reverse, 5′-GCCGGCGTTTGGAGTGGTAGA-3′ (16). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as an internal control. For quantification of mature miRNAs, total RNA was reverse-transcribed using the Taqman miRNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA) and amplified using the TaqMan miRNA assay system (Applied Biosystems). U6 was used as an endogenous control. The relative gene expression was determined by the 2−ΔΔCT method (17).
Plasmids, small interfering RNAs (siRNAs), and miR-423-3p mimic
A fragment containing ZNF674-AS1 was amplified by PCR and cloned to pcDNA3.1(+) expression vector. The p21-targeting siRNA (sip21) was purchased from Invitrogen. miR-423-3p mimic was purchased from Sigma-Aldrich. Two independent siRNAs targeting ZNF674-AS1 were synthesized by Sangon Biotechnology (Shanghai, China), with the target sequences listed as follows: ZNF674-AS1 siRNA#1, 5′-CCTAGATGGCTGTTGTTAT-3′, and ZNF674-AS1 siRNA#2, 5′-ATCTGATGTTAACAGTTGT-3′. Cell transfection was performed using Lipofactamine 3000 transfection reagent (Invitrogen), following the manufacturer’s instruction.
Cells were plated in 96-well plates (5 × 103 cells per well). After culturing for 24-72 h, cells were collected and assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In brief, MTT (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and cultured at 37°C for 4 h. The purple precipitates were dissolved in dimethyl sulfoxide (Sigma-Aldrich). Absorbance was measured at 490 nm.
Colony formation assay
Colony formation assay was performed as described previously (18). Cells were seeded onto 6-well plates (600 cells per well). After incubation for 10 days, cells were fixed and stained with crystal violet (Sigma-Aldrich). The number of colonies per well was counted.
Male BALB/c nude mice, 5 weeks of age, were acclimated to the facility environment (a 12-h light/dark cycle, 23 ± 2°C, and 50% humidity) for 1 week. Xenograft tumors were generated by injecting stably transfected A549 cells (2 × 106) to the flanks of the mice. Tumor volume was measured every week. After 4 weeks mice were euthanatized, and tumors were weighed.
Xenograft tumors were fixed and sectioned. The sections were deparaffinized, probed with anti-Ki-67 antibody (Sigma-Aldrich) in a humidified chamber, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Signals were developed using 3,3′-diaminobenzidine solution (Sigma-Aldrich). The sections were counterstained with hematoxylin.
Cell cycle analysis
For analysis of cell cycle progression, cells were fixed with 70% ethanol and stained with 50 μg/mL propidium iodide (PI) in the presence of 50 μg/mL RNAse A (Sigma-Aldrich). Stained cells were analyzed by flow cytometry.
Western blot analysis
Cells were lysed in ice-cold RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein samples (30 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were incubated overnight at 4°C with the primary antibodies recognizing p21, p27, cyclin D1, CDK4, CDK2, Skp2, and GAPDH. These antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were then incubated with secondary antibodies conjugated to HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Enhanced chemiluminescent reagents (Millipore, Billerica, MA, USA) were used to visualize bound antibodies.
Data are expressed as mean ± standard deviation. Significant differences were analyzed by the Student’s t test or one-way analysis of variance. The relationship of ZNF674-AS1 with clinicopathological parameters was analyzed using the Chi-square test. Survival analysis was performed by the Kaplan-Meier method. Pearson correlation analysis was done to determine the correlation between ZNF674-AS1 and miR-423-3p. P < 0.05 was considered statistically significant.