Sample collection and ethics statement

NSCLC tissues and matched normal adjacent tissues (NATs) were obtained from 50 patients who underwent radical resection of NSCLC in the Thoracic Surgery Department at the First Hospital of China Medical University (Shenyang, China) and had not received any treatment before surgery. All samples were histopathologically confirmed and frozen immediately in liquid nitrogen and stored at − 80 °C. The histological grade was staged according to the Tumor, Node, and Metastasis (TNM) staging Classification. This study was approved by the Research Ethics Committee of China Medical University (Shenyang, China) and was developed according to the principles of the Declaration of Helsinki. Tissue samples were harvested from 50 patients after obtaining their informed consent.

Cell culture

H460, A549, H1975, H1299, HCC-827, SK-MES-1 and 16HBE cell lines were purchased from the Cell Culture Center of Chinese Academy of Medical Sciences (Beijing, China). The PC-9 cell line was purchased from KeyGen Biotech (Nanjing, China). SK-MES-1 cells were maintained in MEM (KeyGen Biotech), while the other cell lines were maintained in RPMI 1640 (KeyGen Biotech). All cell culture media were supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. Cells were cultured at 37 °C in an atmosphere of 5% CO2.

Cell transfection

Cells in the logarithmic growth phase were used for transfection. Cells stably overexpressing PGM5-AS1 (LV-PGM5-AS1) and negative control cells (LV-NC) were generated using lentiviral vectors (GenePharma, Shanghai, China) and screened with puromycin. SLIT2 siRNA, miR-423-5p mimics, miR-423-5p inhibitor and their NC were all purchased from Synbio Tech (Suzhou, China). Transfections were carried out using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

RNA isolation, reverse transcription, and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. Total RNA was reverse transcribed into complementary DNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China).

mRNA and miRNA expression levels were assessed by qRT-PCR using TB Green® Premix Ex Taq™ (TaKaRa) in a final volume of 20 μL containing 10 μL 2 × TB Green Premix Ex Taq, 0.5 μL forward primers, 0.5 μL reverse primers (10 μM), 7 μL nuclease-free water, and 2 μL diluted reverse transcription products. The reaction was amplified using 1 cycle at 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s.

The Bulge-Loop™ miRNA qRT-PCR Primer System (Ribobio, Guangzhou, China) was used to determine the expression levels of miR-423-5p, miR-4709-3p, miR-3065-5p, miR-4766-3p, miR-5701, miR-452-3p, miR-3688 and miR-587, while U6 acted as the internal control. The primer sequences for these miRNAs cannot be listed because they involve company patents.

The other primers used in our study were as follows: PGM5-AS1 forward primer: 5′-TGGTACTTTCAGCCTGTCCG-3′ and reverse primer: 5′- CAGACGGCTTCAGTGGTTGT-3′; SLIT2 forward primer: 5′-GAGAATTTGTCTGCAGTGGTCA-3′ and reverse primer: 5′-AGCTCCAGGAGGGATGACTT-3′; and GAPDH forward primer: 5′-CGGATTTGGTCGTATTGGG-3′ and reverse primer: 5′-CTGGAAGATGGTGATGGGATT-3′ (Sangon Biotech, Shanghai, China).

Subcellular fractionation

Cell nuclear and cytosolic RNA were isolated using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, ON, Canada) according to the manufacturer’s instructions. PGM5-AS1, GAPDH and U6 expression levels in the cytoplasmic and nuclear fractions were determined by qRT-PCR.

Fluorescence in situ hybridization (FISH)

The red FISH Probe of PGM5-AS1 (5′-AUAGUCCCUCUGCCCCGUGCCCU-3′) and FISH kit were purchased from Jijia Biotechnology (Shenyang, Liaoning, China). NSCLC and normal tissue specimens were subjected to FISH assay according to the manufacturer’s instructions.

Cell counting kit-8 (CCK-8) proliferation assay

Cell proliferation was measured using the CCK-8 assay (Beyotime, Shanghai, China). Cells (3.5 × 103 cells/well) were seeded into wells and cultured for 24, 48, 72 and 96 h. At each time point, 10 μL CCK-8 reagent was added to each well and incubated for 90 min at 37 °C. The absorbance was measured at a wavelength of 450 nm using a microplate reader.

Colony formation assay

The colony-forming abilities of H460 and PC-9 cells were measured using the colony formation assay. Cells (600 cells/well) were cultured in 6-well plates for 14 days, then fixed with 4% paraformaldehyde and stained with crystal violet. The colonies in each well were photographed and counted using ImageJ software.

5-ethynyl-2′-deoxyuridine (EdU) assay

Cell proliferation was measured using the BeyoClick™ EdU-555 Kit (Beyotime) according to the manufacturer’s instructions. Images of the EdU-stained cells were captured by fluorescence microscopy and the number of EdU-positive cells were counted using ImageJ software.

Flow cytometry

Cellular apoptosis and the cell cycle were assessed using the Annexin V-APC/PI Apoptosis Detection Kit (KeyGen Biotech) and Cell Cycle Detection Kit (KeyGen Biotech) according to the manufacturer’s instructions, and analyzed by flow cytometry.

Transwell assay

The metastastic ability of the cells was evaluated using Transwell polycarbonate membranes (Corning, New York, USA). For the invasion assay, the upper Transwell chamber was coated with 50 μL diluted Matrigel (BD Bioscience, San Jose, CA) containing 5 μL Matrigel and 45 μL serum-free medium. For the migration assay, the upper chamber was not coated with Matrigel. Cells (5 × 104 cells in 200 μL serum-free medium) were seeded into the upper chamber, while 600 μL medium containing 10% fetal bovine serum was placed in the lower chamber. Cells were incubated for 24 h at 37 °C, then the medium in the upper chamber was removed and cells were fixed with 100% methanol for 10 min. Samples were stained with hematoxylin for 3 min and eosin for 30 s. Non-invading cells were removed from the upper surface of the upper membrane with a cotton swab. The number of cells that had invaded through the membrane were counted in three randomly selected visual fields.

Argonaute2 (AGO2) RNA immunoprecipitation (RIP) assay

The AGO2-RIP assay was performed using the MAGNA RIP® Kit (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s instructions. miR-423-5p and PGM5-AS1 expression levels were analyzed by RT-qPCR.

Luciferase reporter gene assay

Putative lncRNA-miRNA and miRNA-mRNA interactions were verified using luciferase reporter gene assays. The wild-type (WT) PGM5-AS1 (PGM5-AS1-WT) sequence containing miR-423-5p binding sites and its mutant sequences (PGM5-AS1-Mut) were chemically synthesized and inserted into pmirGLO-Report luciferase vectors (Synbio Tech). H460 and PC-9 cells were then co-transfected with the reporter vector and miR-423-5p mimic or mimic NC for 48 h. Luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Western blot analysis

H460 and PC-9 cells were harvested and lysed in lysis buffer containing phosphatase inhibitors, proteinase inhibitor and PMSF (KeyGen Biotech). The protein concentration was determined using the BCA Protein Quantitation Assay Kit (KeyGen Biotech). Protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to PVDF membranes. Membranes were blocked with 5% skim milk, then incubated with a primary antibody against SLIT2 (1:500, ABclonal Technology, Wuhan, China). Following washing, the membrane was then incubated with peroxidase-conjugated affinipure goat anti-rabbit IgG. Protein bands were detected using the GelCapture version software (DNR Bio-Imaging Systems, Jerusalem, Israel). GAPDH (1:1000, Signalway Antibody, Maryland, USA) was used as the internal control for total protein input.

Tumor xenograft model in nude mice

Female nude mice aged 4 weeks (Changsheng Biotech, Benxi, China) were used in this study and maintained under pathogen-free conditions. Stably-transfected H460 cells (5 × 106) were subcutaneously injected into the nude mice. Tumors were measured every 7 days. Mice were humanely killed on day 28 and the tumors were removed. The tumor volume was calculated using the formula: volume = (A × B2)/2, where A is the longest diameter and B is the shortest diameter of the tumor. Tumors were weighed to obtain the tumor weight.

Statistical analysis

Statistical analysis was performed using SPSS 20.0 software (IBM Corp, Armonk, NY, USA).All data are presented as mean value ± standard deviation. Student’s t-test was used to compare the variances between two groups, while one-way ANOVA was used to compare the means of three or more groups. All experiments were performed at least three times independently. A value of P < 0.05 was considered statistically significant.