Clinical specimen and cell culture
Human NSCLC tissues and paracancerous normal lung tissues (N = 43, respectively) were collected from NSCLC patients from the First Hospital of Hunan University of Chinese Medicine. Tissues were restored at − 80 °C. The Ethics Committee of the First Hospital of Hunan University of Chinese Medicine approved this research. The participants signed the written informed consents. The clinicopathologic features of 43 NSCLC patients were shown in Additional file 1: Table S1.
Human lung epithelial cell-line BEAS-2B, NSCLC cell lines (A549 and H1299) and human umbilical vein endothelial cells (HUVEC) were acquired from Procell (Wuhan, China). Cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; Procell), bronchial epithelial cell basal medium (BEBM; Procell) or Ham’s F12K (Procell) at 37 °C with 95% air and 5% CO2. Medium was supplemented with 10% fetal bovine serum (FBS; Procell) as well as antibiotics (100 μg/mL penicillin, 100 μg/mL streptomycin) (Millipore, Bradford, MA, USA). For hypoxia treatment, cells were cultured in a hypoxia incubator chamber (Maworde, Qiqihar, China) with 5% CO2, 1% O2 and 94% N2. PESV, obtained from Chinese Medicine Pharmacy of the First Affiliated Hospital of Hunan University of Chinese Medicine, was used to study the role of circ_0016760 in PESV-mediated NSCLC development and the underlying mechanism.
Ribobio (Guangzhou, China) provided the small interfering RNAs against circ_0016760 (si-circ_0016760#1, 5’-GTCTGGCATGCAGAGGCAGAA-3’; si-circ_0016760#2, 5’-CTGGCATGCAGAGGCAGAAGA-3’ and si-circ_0016760#3, 5’-ATGCAGAGGCAGAAGAGGCCT-3’), the small hairpin RNA targeting circ_0016760 (sh-circ_0016760), miR-29b mimic (miR-29b, 5’-UAGCACCAUUUGAAAUCAGUGUU-3’), miR-29b inhibitor (in-miR-29b, 5’-AACACUGAUUUCAAAUGGUGCUA-3’) and controls (si-con, con, miR-con and in-miR-con). The plasmids overexpressing circ_0016760 (circ_0016760) and HIF1A (HIF1A) were built in Geneseed (Guangzhou, China) using pCD5-ciR and pcDNA vectors.
Si-circ_0016760#2 and circ_0016760 were transfected into cells with controls to reveal the effects of circ_0016760 on PESV-mediated NSCLC process under hypoxia. Si-circ_0016760#2, in-miR-29b, miR-29b or HIF1A was employed to determine the relationships among circ_0016760, miR-29b and HIF1A in regulating NSCLC cell processes under hypoxia. sh-circ_0016760, circ_0016760 and con were employed to demonstrate the impacts between circ_0016760 and PESV treatment on tumor growth in vivo. Cell transfection was conducted using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
TsingZol (Tsingke, Shanghai, China) was used to lyse tissues and cells. RNA isolation reagents (Corning, Madison, New York, USA) were then employed to isolate RNA. Reverse transcription was performed with High-Capacity cDNA Synthesis kits (Thermo Fisher). For detecting the amount of circRNA/miRNA/mRNA, Fast qPCR Mix (Tsingke) was employed. Data were assessed using the 2−∆∆Ct method with U6 and β-actin as controls. Additionally, oligo(dT)18 primers and random primers (Solarbio, Beijing, China) were employed to confirm the circular structure of circRNA. The sense and antisense primers were circ_0016760 5’-AGAGGTTATCCCCATTTTAGAAGTG-3’ and 5’-CATCTGTTCCTGGGTCTGT-3’; HIF1A 5’-GTCTGAGGGGACAGGAGGAT-3’ and 5’-AAAGGCAAGTCCAGAGGTGG-3’; U6 5’-CTCGCTTCGGCAGCACA-3’ and 5’-AACGCTTCACGAATTTGCGT-3’; β-actin 5’-CACCATTGGCAATGAGCGGTTC-3’ and 5’-AGGTCTTTGCGGATGTCCACGT-3’. Ribobio Co., Ltd. provided the primers for miR-29b.
Cell migration and invasion were analyzed by transwell chambers. In short, A549 cells and H1299 cells were mixed with serum-free Ham’s F12K medium (Procell) or RPMI-1640 medium (Procell), and added into the upper chamber, which was coated with Matrigel (Corning) for invasion assay. Ham’s F12K medium and RPMI-1640 medium with 15% FBS (Procell) were severally placed into the lower chamber. At 24 h after transfection of plasmids and oligonucleotides, medium was removed and cells were incubated with methanol (Beyotime, Shanghai, China) and crystal violet (Beyotime). Results were demonstrated via calculating the number of cells from 6 high-power (100x) field microscope (Olympus, Tokyo, Japan).
Cell colony formation assay
The colony-forming ability of A549 and H1299 cells was revealed by colony formation assay. Shortly, cells (500 cells per well) were grown in 6-well plates for 2 weeks after transfection of plasmids and oligonucleotides, and Ham’s F12K medium and RPMI-1640 medium (Procell) were replaced every 3 days. Proliferative colonies were immobilized and stained with paraformaldehyde (Beyotime) and crystal violet (Beyotime), respectively. Cell colony-forming ability was analyzed by determining the number of colonies.
3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cells were grown in 96-well plates for 16 h, and treated with test compounds. At 48 h after transfection, cells were went through 4 h incubation with MTT solution (Beyotime). After that, dimethyl sulfoxide was placed into each well. Samples were assessed via detecting the absorbance at 490 nm with microplate reader (REAGEN, Shenzhen, China).
Tube formation assay
Angiogenic capacity of cells was analyzed by capillary-like network formation assay as descripted previously . In brief, the cells were passaged in 96-well microplates coated with growth factor-depleted Matrigel (Corning). After 16 h of culture in NSCLC-conditioned medium, the branch points containing at least 3 cells were counted under microscope (Zeiss, Melville, NY, USA). Results were analyzed using image J software.
Flow cytometry analysis
Cell apoptotic rate was analyzed using Annexin V-FITC and propidium iodide apoptotic detection kit (Solarbio). In brief, the cells were harvested after various treatments, and suspended in Binding buffer. Then, Annexin V-FITC and propidium iodide were used to incubate the cells in the dark. Finally, cell apoptotic rate was analyzed using a flow cytometer with CytExpert software.
Dual-luciferase reporter assay
The binding sequences between miR-29b and circ_0016760 or HIF1A were predicted by starbase online database and microT CDS online database, respectively. The wild-type (WT) plasmids of circ_0016760 (circ_0016760-WT) and HIF1A (HIF1A-WT) and their mutant (MUT) plasmids (circ_0016760-MUT and HIF1A-MUT) were constructed by Ribobio Co., Ltd. Cell transfection was performed according to the literature’s methods . Luciferase activities were detected using a Dual-Lucy Assay Kit (Solarbio).
RNA immunoprecipitation (RIP) assay
In short, cells were harvested and lysed using RIP lysis buffer (Millipore). Lysates were incubated with magnetic beads coated with anti-argonaute2 (anti-Ago2; Abcam, Cambridge, UK) or anti-immunoglobulin G (anti-IgG; Abcam) for 24 h. The expression of circ_0016760, miR-29b and HIF1A enriched by anti-Ago2 or anti-IgG was detected by qRT-PCR.
Western blot analysis
The lysates from cells and tissues were acquired by using RIPA buffer (Beyotime), and then loaded onto 12% SurePAGE gels (Thermo Fisher). Protein bands were electrotransferred onto polyvinylidene fluoride membranes (Millipore) prior to blocking aspecific signals using 5% nonfat milk (Solarbio). After that, the membranes were incubated with primary antibodies and secondary antibody (1:8000; Affinity, Nanjing, China), respectively. Protein bands were visualized using RapidStep ECL Reagent (Millipore). β-actin was employed as a reference. Primary antibodies were anti-HIF1A (1:1500; Affinity), anti-proliferating cell nuclear antigen (anti-PCNA; 1:800; Otwo Biotech, Shenzhen, China) and anti-β-actin (1:1000; CST, Boston, MA, USA).
In vivo tumor formation assay
Vital River Laboratories (Beijing, China) provided male BALB/c nude mice (5-week old). Nude mice were divided into 4 groups (N = 6, respectively): con group, sh-circ_0016760 group, PESV group and PESV + circ_0016760 group. 5 × 106 A549 cells transfected with sh-circ_0016760, con or circ_0016760 were hypodermically injected into the mice. Mice were intragastrically administered with PESV (200 mg/kg) once a day from tumor formation to the end of experiments. On the 7th day after injection, tumor volume was monitored every 7 days. Twenty-eight days later, mice were administrated with xylazine (10 mg/kg; Seebio Biotech, Shanghai, China) and then euthanized by cervical dislocation. The forming tumors were excised for the analysis of tumor weight and gene expression. The Animal Care Committee of the First Hospital of Hunan University of Chinese Medicine agreed with this study.
Immunohistochemistry (IHC) assay
IHC assay was performed on the primary tumors from A549 cells according to the standard method . In brief, 4-µm-thick sections embedded into paraffin were heated and deparaffinized with xylene. The slides were incubated with H2O2 and primary antibodies specific to HIF1A (1:200; Cusabio Biotech, Wuhan, China), proliferating cell nuclear antigen (Ki67; 1:200; Cusabio Biotech) and Cleaved Caspase-3 (1:200; Cusabio Biotech). Subsequent steps were carried out using IHC assay kit (Phygene, Fuzhou, China) as instructed. CX31-LV320 microscope (Olympus) was utilized to capture images. The relative expression of the three proteins was calculated based on the percentage of stained cells and intensity of immunostaining as instructed .
SPSS 21.0 software was employed to analyze the data from 3 independent duplicate tests. Results were shown as means ± standard deviations (SD). Significant differences were demonstrated by Student’s t-tests, Wilcoxon rank-sum test or one-way analysis of variance. P value < 0.05 was considered statistically significant.