Non-small cell lung cancer tissues (n=50) and the neighboring normal lung tissues (n=50) were collected from 50 NSCLC patients who underwent surgery at Shanghai University of Traditional Chinese Medicine hospital. All NSCLC patients involved in this study gave the signed informed consents. The experiments in the present research were approved by the Human Research Ethics Committee of Shanghai University of Traditional Chinese Medicine hospital. None of the NSCLC patients participating in this research had undergone other therapy.
Two NSCLC cell lines (A549 and H1299) were obtained from American Type Culture Collection (Manassas, VA, USA), and the human bronchial epithelial cell line (16-HBE) used in this study was purchased from Shanghai Fuxiang Biological Technology Co. Ltd. (Shanghai, China). 16-HBE and NSCLC cells were cultured in modified Eagle’s medium (MEM; Gibco, Rockville, MD, USA) and Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), and streptomycin/penicillin (100 U/mL; Invitrogen), respectively. All cells were maintained at 37˚C in a humidified incubator with an atmosphere of 5% CO2.
Small interfering RNA (siRNA) against circ_0018534 (si-circ_0018534) and the negative control (si-NC), miR-153-3p mimic (miR-153-3p) and the negative control (miR-NC), miR-153-3p inhibitor (anti-miR-153-3p) and its control (anti-miR-NC), the overexpression plasmid of BTBD7 (pcDNA-BTBD7) and the corresponding control (pcDNA), small hairpin RNA targeting circ_0018534 (sh-circ_0018534) and control (sh-NC) were built by Genepharma (Shanghai, China) and were transfected into cells using Lipofectamine 2000 Reagent (Invitrogen).
Haematoxylin and eosin (HE) staining
Tumor tissues from each group of collected NSCLC sufferer specimens were cut into the 5-μm sections, and immobilized with paraformaldehyde (Sigma, Billerica, MA, USA). Then, the section was dehydrated using with ethanol (Millipore, Bradford, MA, USA) and embedded in paraffin. HE (Beyotime, Shanghai, China) was employed to stain the tissues. The images were captured under a microscope (Nikon, Tokyo, Japan).
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from NSCLC tissues and cells using Trizol reagent (Invitrogen). TaqMan™ Reverse Transcription reagent (Thermo Fisher Scientifc, Inc., Waltham, MA, USA) was used to transcribe RNA to complementary DNA. The expression of circ_0018534, miR-153-3p and BTBD7 was analyzed by an ABI 7500 Real-time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). U6 was used as the internal control for miR-153-3p, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was applied as the internal load for circ_0018534 and BTBD7. We designed specific primers for miR-153-3p (F: 5’- ACACTCCAGCTGGGTTGCATAGTCACAAAA-3’, R: 5’-TGGTGTCGTGGAGTCG-3’), circ_0018534 (F: 5’-AGGTGGTTTCTTTGGATT-3’, R: 5’-CCATGTCAAGAGGGTAAG-3’), BTBD7 (F: 5’-TGCGAATGGTTAGAATGT-3’, R: 5’-GAGACGAGGAACTGGAAT-3’), U6 (F: 5’- TCCGATCGTGAAGCGTTC-3’, R: 5’- GTGCAGGGTCCGAGGT-3’), GAPDH (F: 5’-TGAACGGGAAGCTCACTGG-3’, R: 5’-TCCACCACCCTGTTGCTGT-3’). The data were calculated using the 2 -∆∆Ct method.
Total protein was extracted from NSCLC tissues and cells by using RIPA lysis buffer (Beyotime). Then 30 μg total protein was applied and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein. After loading for 2 h, the protein was transferred to membranes (Sigma) and 5% milk was used to block the membranes. Then the membranes were incubated with the primary antibodies against BTBD7 (1:500, ab121006, Abcam, Cambridge, UK), matrix metalloprotein 9 (MMP9) (1:5000, ab76003, Abcam), Snail (1: 1000, ab216347, Abcam), B-cell lymphoma-2 (Bcl-2) (1:2000, ab182858, Abcam), BCL2-associated x protein (Bax) (1:5000, ab32503, Abcam), proliferating cell nuclear antigen (PCNA) (1:1000, ab265609, Abcam) and β-actin (1:1000, ab8226, Abcam) overnight. Secondary antibodies included goat anti-rabbit IgG H&L (1:5000, ab6741, Abcam) and goat anti-mouse IgG H&L (1:2000, ab205719, Abcam). Finally, an enhanced chemiluminescence kit (GE Healthcare, Waukesha, WI, USA) was used to detect the immunoreactive proteins.
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay
The viability of NSCLC cells was measured by MTT assay. After transfection, NSCLC cells (5×103) in 96-well plates were cultured for 24 h, 48 h and 72 h. MTT reagent was added into the medium for 4 h. Subsequently, the medium was removed, and dimethyl sulfoxide was added to dissolve the blue crystal. Finally, the absorbance was detected at 450 nm by a microplate reader (Bio-Rad Laboratories).
5-Ethynyl-29-deoxyuridine (EdU) assay
The proliferation of A549 and H1299 cells was determined by EdU assay kit (Ribobio, Guangzhou, China). In brief, cells were grown in 12-well plates after transfected with plasmids or oligonucleotides. At 48 h after culture, cells were digested and seeded in 96-well plates. When the confluence of cells was about 30%, the EdU assay was carried out according to the instruction of manufacture. Immunostainings were visualized and captured with a fluorescence microscope (Olympus, Tokyo, Japan).
Cell apoptosis assay
Cell apoptosis was detected by flow cytometry analysis. The treated HSCLC cells were collected and centrifuged at 1000 rpm for 8 min. Then phosphate buffered solution (PBS) was used to wash the treated NSCLC cells, and the binding buffer was used to resuspend cells. Subsequently, cells were stained using Annexin V/Propidium Iodide (BestBio, Shanghai China) for 20 min in the dark. Cell apoptosis was measured by flow cytometry (Becton-Dickenson, Franklin Lakes, NJ, USA).
Transwell migration and invasion assay
The transwell chambers used in cell migration and invasion assays were purchased from Corning Incorporated (Big Flats, NY, USA). The chambers pre-coated or non-coated with Matrigel (BD Biosciences, San Jose, CA, USA) were used for cell invasion or migration assay, respectively. In the upper chamber, there were the cells mixed with serum-free medium, while DMEM containing 10% FBS was added into the lower chamber. Then cells not migrated or invaded were scraped, whereas the cells removed to the lower chamber were fixed, stained, and analyzed.
Dual-luciferase reporter assay
The circ_0018534 sequence and the 3’UTR fragment of BTBD7 containing the putative miR-153-3p binding sites were amplified by polymerase chain reaction and subsequently inserted into pGL3 promoter vector (Invitrogen), termed as circ_0018534-wild-type (circ_0018534-WT) and BTBD7-wild-type (BTBD7 3’UTR-WT). And the mutant sequences of circ_0018534 and BTBD7 were used to establish circ_0018534-mutant (circ_0018534-MUT) and BTBD7-mutant (BTBD7 3’UTR-MUT) reporter vectors. Then A549 and H1299 cells were co-transfected with reporter vectors with miR-153-3p mimics or miR-NC. The luciferase activity was assessed by the dual-luciferase reporter assay system (Promega, Madison, WI, USA).
Xenograft mouse model
BALB/c nude mice were purchased from the Beijing Laboratory Animal Center (Beijing, China). The mice were housed in a standard animal laboratory with a 12 h light-dark cycle, and free access to food and water. A549 cells (2 × 106) stably transfected with short hairpin RNA (shRNA) against circ_0018534 (sh-circ_0018534) or sh-NC were subcutaneously injected into the right flank of nude mice (5 per group). The transfected nude mice were classified into two groups: sh-NC nude mice group and circ_0018534 knockdown nude mice group. After 7 d, the tumor volume was detected for the first time after the tumors became visible. Subsequently, the volume in every group was measured using a caliper every 4 d. After injection for 28 d, all the mice were euthanized by asphyxia method. The mice were treated with the flow rate of CO2 to displace 60% air of the chamber volume/min according to the current guideline of American Veterinary Medical Association (AVMA). The tumor weight was determined. The lung tissues were separated from the nude mice and stored at -80˚C. The experiments were approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine hospital.
All data were displayed as the means ± the standard deviations (SD). SPSS 18.0 software was used to analyze the data in the present study. Spearman correlation analysis was performed to analyze the correlation between variables. Student’s t-test was used to analyze the statistical difference between groups. One-way analysis of variance (ANOVA) was compared the statistical difference among three or more groups. A value of P < 0.05 meant a statistically significant difference.