All specimens were obtained from the Department of Thoracic Surgery, Hunan Provincial People’s Hospital. A total of 132 pairs NSCLC tissues and compared normal tissues were confirmed pathologically. More specifically, tumor and adjacent normal tissues were obtained from the patients who were diagnosed with NSCLC and underwent initial surgery between September 2014 and June 2016. These patients had not received any systemic treatment, including radio-chemical therapy, before sampling. The protocol was approved by the ethical review committee of Hunan Provincial People’s Hospital, and patient consent was obtained before the samples were taken.
Cell line culture and transfection
The human NSCLC cell lines (A549 cells and HCC827 cells) were provided by the American Type Culture Collection (ATCC, Rockville, MD). Cells received incubation treatment in RPMI-1640 (Solarbio, Beijing, China) and dulbecco’s modified eagle medium (DMEM; Solarbio) composed of 1% penicillin/streptomycin (Solarbio) and 10% fetal bovine serum (FBS; Solarbio) under 5% CO2 at 37 °C. A pcircRNA2.2-circ_0009043 overexpression plasmid and empty pcircRNA2.2 plasmid, the pcDNA 3.1 plasmid specific to DNAJB4 (termed OE- DNAJB4) and empty pcDNA3.1 plasmid were purchased from Guangzhou RiBoBio Co., Ltd. (Guangzhou, China). Circ_0009034 short hairpin (sh) RNA (shcirc) (AATTCAAAAAACCATGAAGCAAAATCAAGTGATCTCTTGAAGTAGCACAACATTCTCCACCCG), scrambled shRNA (shCTRL), DNAJB4 short hairpin (sh) RNA (shDNAJB4) (AATTCAAAAAACAGAAGCTTATGAAGTATTGAGTTCTCTTGAAGTAGCACAACATTCTCCACCCG), miR-148a-3p mimics, NC miRNA mimic (mimic NC), miR-148a-3p inhibitor, and inhibitor NC were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was employed to transfect abovementioned plasmids into A549 cells and HCC827 cells. 48 h after the transfection, we harvested cells for later study.
Cell proliferation experiment
The proliferation of HCC cells was tested through Cell Counting Kit-8 (CCK-8) assay, the transfected A549 and HCC827 cells (1 × 104 cells/well) were added into 96-well plates and maintained for 48 h. Next, 10 μL CCK-8 reagent (Sigma-Aldrich) was added into each well and cultured for further 2 h. After that, a microplate reader was adopted to examine the optical density value at 450 nm.
Cell proliferation was also assessed using an EDU assay kit (Ribobio, Guangzhou, China). For this purpose, after seeding 1 × 106 cells into each well of confocal plates, a 2-h incubation was performed at 37 °C with 50 μM of EDU buffer. Cell fixing was then carried out for half an hour using 4% of formaldehyde before cell permeabilization for 20 min using 0.1% Triton X-100. Eventually, after adding the EDU solution, cell nuclei were stained with Hoechst prior to visualization under a fluorescence microscope.
In vivo xenograft experiments
Nude female mice from Vital River Laboratories (Beijing, China) were housed under specific pathogen-free conditions in the Department of Laboratory Animal Science of Hunan Provincial People’s Hospital. The Ethics Review Committee of the Department of Laboratory Animal Science of Hunan Provincial People’s Hospital approved this study. All animals were randomly assigned to five groups (n = 6/group) that were then subcutaneously implanted in the left flank with A549 cells (1 × 107) that were either WT cells or cells that had been stably transfected with the pcircRNA2.2, pcircRNA2.2-circ_0009043 vector, NC‑shRNA, or circ_0009043‑shRNA constructs. Tumor volume was measured once per week using calipers (V = length × width2 × 0.5) for four weeks, after which mice were euthanized. Tumors were then excised, fixed for 24 h using 4% paraformaldehyde, stained via TUNEL assay, and subjected to downstream immunohistochemical staining for DNAJB4, followed by imaging with a brightfield microscope (Olympus, Tokyo, Japan).
Quantitative real-time reverse transcription PCR
Tissue samples and cells were collected, and total RNA was extracted using the TRIzol reagent (Invitrogen, Waltham, MA, USA) for quantitative real-time reverse transcription PCR (qRT-PCR) detection. The cDNA obtained by reverse transcription using the Promega GoScript reverse transcription system (Promega Madison, WI, USA) was subjected to quantitative real-time PCR in a 20 µL PCR amplification system using the ABI7500 real-time quantitative PCR instrument. For miRNAs, miRNA first-strand cDNA was constructed using the stem-loop method  using the cDNA Synthesis Kit (R601, Novabio, Shanghai, China). Stem-loop primer 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAAAGTT-3’ was purchased from General Biol (Anhui, China). The differences in the expression levels of different genes were analysed using 2−ΔΔCt relative quantification. The primers were circ_0009043: (F: 5’-TCCGCAAACATTCAGACAAA-3’ and R: 5’-GCTTCAGCTCTTCCATTGCT-3’); miR-148a-3p: (F: 5’-ACACTCCAGCTGGGTCAGTGCACTACAGAACT-3’ and R: 5’-CTCAACTGGTGTCGTGGA-3’); DNAJB4: (F: 5’-TGGGGACGCTGTTTTCTTTTAC-3’ and R: 5’-CTCCTGCTCCTCCTTTCAACC-3’); U6: (F: 5’-CTCGCTTCGGCAGCACA-3’ and R: 5’-AACGCTTCACGAATTTGCGT-3’); and GAPDH: (F: 5’-CTCCTCCTGTTCGACAGTCAGC-3’ and R: 5’-CCCAATACGACCAAATCCGTT-3’).
After isolation from tissue or cell samples, total proteins were separated via SDS-PAGE and transferred onto PVDF membranes that were blocked with an appropriate solution for 1 h at room temperature. Blots were then probed for 2 h at room temperature with primary antibodies specific for Bax (1:1000, BA0315-2, Boster), Bcl-2 (1:1200, A00040-1, Boster), Cytochrome C (1:5000, ab133504, Abcam), DNAJB4 (0.1 µg/ml, ab254641, Abcam), and GAPDH (1:10,000, BA2913, Boster). Blots were then probed for 1 h at room temperature using Goat Anti-Rabbit IgG H&L (HRP) (1: 2000, ab6721, Abcam), after which they were washed, incubated in developer solution, and exposed to X-ray film to detect protein bands.
Nuclear and cytoplasmic fractions from A549 and HCC827 cells were isolated using a PARIS Kit (Invitrogen) based on provided directions. Circ_0009043 abundance in these two fractions was assessed via qPCR, with U6 and GAPDH being used as respective normalization controls for nuclear and cytoplasmic transcripts.
Dual-luciferase reporter gene assay
Potential miRNA binding partners for circ_0009043 were predicted using an online tool (https://circinteractome.nia.nih.gov/). Luciferase reporter constructs were then prepared for circ_0009043 and miRNAs of interest, with HEK293T cells being transfected using WT or mutant circ_0009043 reporter constructs and transfected with appropriate miRNA mimic or control constructs. At 48 h post-transfection, firefly and Renilla luciferase activity was assessed, with results being reported as the ratio of these two activity levels.
RNA immunoprecipitation (RIP)
A Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) was used based on provided directions. Total RNA and species-appropriate IgG (n = 3) were utilized as control samples to confirm the specificity of the detected RNA signal. For the anti‐AGO2 RIP assay, NSCLC cells were transfected using miRNA mimics after which RIP was performed using anti-AGO2 (2 µg/mL, clone number: ab32381; Abcam) at 48 h post-transfection.
After incubation for 48 h, cells that have been transfected and were seeded for the counting in the 96-well plates were then removed from the plates by using trypsin reagent in the absence of ethylenediaminetetraacetic acid, and cells were then rinsed with cooled PBS solution. After that, centrifugation was done, and the PBS solution was discarded. Then annexin V–fluorescein isothiocyanate (FITC) apoptosis discovery kit (BioLegend, Inc.) was applied to detect the status apoptotic of the obtained cells. Resuspension of cells was then carried out in 100 μL of buffer (1 × binding buffer). Then resuspended cells were further treated with 5 μL propidium iodide and annexin V–FITC in the darkness for the 15-min duration, and at room temperature. Finally, for the analysis of the double-stained cells, the FACSCalibur Flow Cytometry instrument (BD Biosciences, San Jose, CA, USA) was used. Version 2.9 of the CellQuest (BD Biosciences) was used for the evaluation of the proportion of apoptotic cells.
Immunohistochemical (IHC) staining
Tissue sections were deparaffinized, subjected to antigen retrieval, treated to quench endogenous peroxidase activity, blocked using goat serum (Gibco), and incubated with primary anti-DNAJB4 (1 µg/ml, ab254641, Abcam) and secondary antibodies (abcam; #ab205718; 1:1000). Sections were then imaged using a phase-contrast microscope (Leica, Cat. #DMI 1).
Terminal deoxynucleotidyl transferase-medimed dUTP nick-endlabeling (TUNEL) assay
A one-step TUNEL kit (C1089, Beyotime Institute) was used as in prior reports to assess apoptotic cell death. Briefly, NSCLC tissue Sect. (3 μm) were deparaffinized, treated for 30 min with DNase- protease K (20 μg/mL) at 37℃, rinsed using PBS, and stained in the dark for 1 h with 50 μL of TUNEL reaction mixture at 37℃. Sections were then rinsed using PBS and imaged with a fluorescence confocal microscope (Zeiss LSM710, Germany). TUNEL-positive cells in 10 random fields of view in size sections were then counted using Image J (Bio-Rad Laboratories, CA, USA).
SPSS 18.0 (SPSS, Inc.) was used to analyze all data. Data were compared between groups using t-tests and one-way ANOVAs with Dunnett’s post hoc test as appropriate. Pearson’s correlation coefficient was used to assess the relationships between circ_0009043, miR-148a-3p, and DNAJB4 expression. Data are given as means ± SD from triplicate experiments, with P < 0.05 as the significance threshold.