The clinical data for LAC tissues as well as the relative expression levels of EIF4A3, Wnt10b and miR-885-3p were downloaded from The Cancer Genome Atlas (TCGA) RNA-seq database (https://genome-cancer.ucsc.edu). A tissue microarray including 90 paired LAC tissues was purchased from Superbiotek Pharmaceutical Technology (Shanghai, China). The specimens were classified according to the TNM staging, and diagnosed by two independent pathologists. Our study was approved by the Ethics Committee of Zhengzhou University.
CircRNA expression profiling
Total RNA from LAC and adjacent normal tissues (n = 3) was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were conducted according to the Arraystar’s standard protocols. The detailed description of this process was carried out according to the previous report .
Identification of circTUBGCP3-specific binding with miRNAs
CircTUBGCP3 specific binding with miRNAs (miR-885-3p, miR-640, miR-324-5p, miR-194-3p and miR-103a-3p) was identified by circRNA expression profiling and miRbase. The targets of miR-885-3p were identified by TargetScan7.1 (http:// www.targetscan.org/vert_71/).
Normal pulmonary epithelial cells BEAS-2B and LAC cell lines (A549, NCI-H23, NCI-H1993, SPC-A1, NCI-H460) were stored in liquid nitrogen in our hospital. They were cultured in Dulbecco’s Modified Eagle medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 µg/ml of streptomycin (HyClone) in a humidified atmosphere containing 5% CO2 at 37℃.
Fluorescence in situ hybridization (FISH)
Digoxin-labeled probe sequences for circTUBGCP3 (5'-GGATCACATCATTGC TGCAC-3') were used for analysis of the expression and localization of circTUBGCP3 in LAC tissues and cells. The detailed description of FISH analysis was conducted as previously reported . The analysis software Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to obtain the immunofluorescence accumulation optical density of circTUBGCP3 in LAC tissues.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted by using TRIzol, reverse transcription was performed by using M-MLV and cDNA amplification by using the SYBR Green Master Mix kit (Takara, Otsu, Japan). Total RNA was isolated using a High Pure miRNA isolation kit (Roche) and RT-PCR using a TaqMan MicroRNA Reverse Transcription kit (Life Technologies). The primers were listed in Table S1.
Western blot analysis
LAC cell lines (NCI-H460 and A549) were harvested and extracted by using lysis buffer. Cell extracts were boiled in loading buffer and equal amount of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride membranes. The primary antibodies anti-Wnt10B (ab70816, Abcam, USA), anti-β-catenin (ab2365, Abcam, USA) and anti-GAPDH (#5174, CST, Shanghai, China) were diluted at a ratio of 1:1000 according to the instructions and incubated overnight at 4℃. The detailed description of Western blot analysis was performed as previously reported .
Plasmid, siRNA, miRNA mimic and inhibitor
Plasmid-mediated circTUBGCP3 vectors, lentivirus-mediated siRNA targeting circTUBGCP3 vector (si-circTUBGCP3, 5’-GACAGTGACTCCAGGTTTTTT-3’) and miR-885-3p mimic/inhibitor and EIF4A3 plasmids were purchased from GenePharma (Shanghai, China). The negative controls such as NC, si-NC, pEX-3 or miR-NC was used as the control vectors. NCI-H460, A549 and SPC-A1 cell lines were planted in 6-well plates 24 h prior to si-circTUBGCP3, circTUBGCP3, miR-885-3p mimic or inhibitor transfection with 50–60% confluence, and then were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture instructions.
Luciferase reporter assay
NCI-H460, A549 and SPC-A1 cell lines were seeded into 96-well plates and were co-transfected with PRL-TK-pMIR-circTUBGCP3 or PRL-TK-pMIR-Wnt10b 3’UTR, and miR-885-3p mimic/inhibitor or miR-NC. After 48 h of incubation, the firefly and Renilla luciferase activities were detected with a dual-luciferase reporter assay (Promega, Madison, WI, USA).
MTT, colony formation, EdU and Transwell assays
MTT, colony formation, 5-ethynyl-2-deoxyuridine (EdU) and Transwell assays were performed as previously reported .
RNase R and Actinomycin D treatment
Total RNA (2µg) was incubated for 30 min at 37℃with 3 U/µg of RNase R (Epicentre Technologies, Madison, WI, USA). Transcription was prevented by the addition of 2 mg/ml Actinomycin D or DMSO (Sigma-Aldrich, St. Louis, MO, USA) as the negative control. After A549 and SPC-A1 cell lines were exposed to RNase R or Actinomycin D treatment, the enrichment levels of circTUBGCP3 and TUBGCP3 were detected by qPCR analysis.
RNA immunoprecipitation (RIP)
RIP assay was performed in A549 and SPC-A1 cell lines by using a Magna RIP RNA-binding protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Antibodies for RIP assays against Ago2 (ab5072) and IgG were purchased from Abcam, USA.
In vivo tumorigenesis assay
Male nude mice (6 weeks old) were purchased from Shanghai SIPPR-BK Laboratory Animal Co. Ltd (Shanghai, China) and maintained in microisolator cages. All the animals were conducted according to the institutional guidelines, and approved by the Animal Ethics Committee of Zhengzhou University. The mice were subcutaneously inoculated with 1×107 of NCI-H460 cells stably transfected with si-circTUBGCP3 or si-NC. The body weight and tumor size were measured every other day, and the tumor volume was obtained according to the formula: length × width2/2.
Statistical analyses were conducted by using SPSS 20.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism. Student’s t-test or Chi-square test was used to analyze the statistical data between two groups, but Analysis of Variance was used to estimate the statistical significance for comparisons of more than three groups. Overall survival curves were analyzed with the Kaplan-Meier method and log-rank test. Univariate analysis and multivariate models were performed by using a Cox proportional hazards regression model. P < 0.05 was considered statistically significant.