The expression of circ_0001361 was determined in 32 pairs of LUAD tissues and matched non-cancerous lung tissues collected from patients with informed consents from Geriatric Hospital of Nanjing Medical University between January 2017 and January 2019. Chemotherapy or radiotherapy was not given to these patients before surgery. All experiments were permitted by the Ethics Committee of Geriatric Hospital of Nanjing Medical University.
Cell lines and cell culture
Normal human bronchial epithelial (HBE) cells, A549, H1975, PC9, and Calu-3 cells were obtained from the cell bank of Cancer Research Institute of Central South University (Hunan, China). All cells received routine mycoplasma test were cultured in RPMI-1640 supplemented with 10% fetal bovine serum at 37 °C containing 5% CO2.
The expression plasmid of VMA21 was constructed by cloning cDNA sequences into pcDNA3.1 vector. The circ_0001361 overexpression plasmid was obtained from Geenseed Biotech (Guangzhou, China) . pGPU6-shRNA targeted circ_0001361 (sh-circ_0001361), pGPU6-shRNA negative control (sh-NC), sh-VMA21, miR-525-5p mimics, mimics negative control (mimic NC), miR-525-5p inhibitor, and inhibitor NC were synthesized by Shanghai GenePharma Co., Ltd. Transfection of the above plasmids and oligonucleotides was carried out using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). Stable transfected cells were selected using puromycin after the transient transfection and then were utilized in subsequent experiments.
Preparation of nuclear and cytoplasmic RNA
The nuclear and cytoplasmic RNA was extracted using a PARIS Kit (Thermo Fisher). In brief, cells were lysed and centrifuged to obtain the nuclear and cytoplasmic fractions, followed by adding with 2× Lysis/Binding Solution and 100% ethanol. Then the mixture was filtered and eluted before subsequent detection.
TRIzol reagent (Thermo Fisher) was adopted for the isolation of total RNA from tissues and cells. Subsequently, cDNA was generated using PrimeScript RT Master Mix (Takara, Osaka, Japan) with random oligo for circRNA and mRNA, or using miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, Beijing, China) for miRNA. qRT‐PCR reactions were conducted using SYBR Green Realtime PCR Master Mix (SinoBio, Shanghai, China) for circRNA and mRNA, or miRcute miRNA qPCR Detection Kit (TIANGEN) for miRNA. PCR primers were as follows: circ_0001361: 5′‐GAGATGCAGCTCAGCAGGTTA‐3′ (forward) and 5′‐AATGGTGGCAGTTCCAGAGG‐3′ (reverse); VMA21: 5′‐AGACGCTCCTGTTCTTCACA‐3′ (forward) and 5′‐CATACACAAAGAGGGCCAGC‐3′ (reverse); 18S rRNA: 5′‐CTTGGTCATTTAGAGGAAGTAA‐3′ (forward) and 5′‐GCTGCGTTCTTCATCGATGC‐3′ (reverse); miR-525-5p: 5′‐GGCTCCAGAGGGATGCA‐3′ (forward) and 5′‐GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGAAAG‐3′ (reverse); U6: 5′‐GCTTCGGCAGCACATATACTAAAAT‐3′ (forward) and 5′‐CGCTTCACGAATTTGCGTGTCAT‐3′. (reverse). Circ_0001361 and VMA21 expression levels were normalized to 18S rRNA, and miR-525-5p expression was normalized to U6. 2−ΔΔCT method was used for the analysis of data.
Cell proliferation assay
To assess the proliferation of A549 and PC9 cells, MTT assay was adopted. After incubation for the indicated time points, A549 and PC9 cells in 96-well plates were added with MTT (5 mg/mL, Amresco, Solon, OH, USA) and maintained at 37 °C for 4 h, followed by dissolving in 100 μL of dimethylsulfoxide (DMSO). The absorbance at 490 nm was detected on a microplate reader (Thermo Fisher).
Colony formation assay
A549 and PC9 cells subjected to various transfections were seeded in 6-well plates (1000 cells per well) and cultured for 14 days. Then the cells were immersed in 0.1% crystal violet (Beyotime, Haimen, China) for staining and imaged under a microscope (Olympus, Tokyo, Japan).
Transwell invasion assay
The invasion ability of A549 and PC9 cells was detected by Transwell chambers (8 μm, Corning, Tewksbury, MA, USA) with Matrigel matrix (Corning). Briefly, the cells resuspended in serum-free medium were placed into the upper chambers. The lower chambers were added with culture medium containing 10% fetal bovine serum. After incubating in a 37 °C incubator for 24 h, the invaded cells were stained with 0.1% crystal violet, photographed and counted.
Wound scratch assay
A549 and PC9 cells were seeded in 24-well plates. After reaching full confluence, a scratch was made using a 200-µl pipette tip. The floating cells were removed by washing with phosphate-buffered saline. After incubation in serum-free medium for 24 h, the migrated cells were photographed and quantitatively analyzed.
Dual luciferase assay
The predicted interaction between miR-525-5p and circ_0001361/VMA21 was verified by dual luciferase assay. The sequences of circ_0001361/VMA21 containing the predicted or mutant binding sites for miR-525-5p was inserted into pmirGLO vector (Promega, Madison, WI, USA). The constructed plasmids circ_0001361/VMA21-wild type (WT) containing miR-525-5p binding sites or circ_0001361/VMA21-mutant (MUT) containing mutant miR-525-5p binding sites together with miR-525-5p mimics or mimic NC were co-transfected into A549 cells. The luciferase activity in cells after the transfection for 24 h was evaluated using Dual-Lucy Assay Kit (Solarbio, Beijing, China).
RNA immunoprecipitation (RIP) assay
The combination between circ_0001361 and miR-525-5p was determined by RIP assay using an Imprint RNA Immunoprecipitation kit (Merck Millipore, Billika, MA, USA). Briefly, the collected cells were suspended in lysis buffer. After centrifuging at 12,000 g for 30 min, Ago-2 antibody or IgG antibody pre-bound to magnetic beads was added to cells. After incubation over night at 4 °C, RNA was eluted and RT-qPCR was carried out to assess the expression of circ_0001361.
RNA pull-down assay
For RNA pull-down assay, biotinylated WT miR-525-5p (bio-miR-525-5p-WT) or mutant miR-525-5p (bio-miR-525-5p-MUT) was transcribed into the cells. After the transfection for 48 h, the cells were collected and suspended in IP Cell lysis Buffer (Sangon, Shanghai, China) containing protease inhibitor cocktail for 10 min on ice. Subsequently, biotin-coupled RNA complex was purified after the incubation with streptavidin-coated magnetic beads (Thermo Fisher) at 4 °C for 3 h. The abundance of circ_0001361 was determined by RT-qPCR.
The tissues and cells were lysed in RIPA buffer (Solarbio) supplemented with protease inhibitor cocktail (Solarbio). After protein quantification using BCA protein assay kit (Solarbio), the lysates with equal amount protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were immersed in 5% skim milk for 1 h, and then probed with the antibodies against VMA21 (1:500, 21921-1-AP, Proteintech, Wuhan, China) and GAPDH (1:5000, 10494-1-AP, Proteintech) overnight at 4 °C. Then HRP-conjugated Goat Anti-Rat IgG (1:2000, SA00001-15, Proteintech) was applied to the membranes. The membranes were visualized using ECL Western Blotting Substrate (Solarbio).
Mouse xenograft model
Male BALB/c nude mice were purchased from Hunan Slac Jingda Laboratory Animal Co., Ltd. (Hunan, China). A549 and PC9 cells infected with lentivirus expressing sh- circ_0001361 or sh-NC (Genechem, Shanghai, China) were subcutaneously inoculated into the mice. The tumor size was measured every 4 days and the volume of tumors was calculated as following: volume = (length × width2)/2. All mice were euthanized at 5 weeks after the inoculation. The tumors were resected, weighed, and the expression levels of circ_0001361, miR-525-5p, and VMA21 were assessed by RT-qPCR and Western blotting. All animal experiments were approved by the ethics Geriatric Hospital of Nanjing Medical University.
Data from at least three independent experiments are shown as mean ± standard deviation (SD). Student’s t test or one-way analysis of variance followed by Bonferroni’s multiple comparison test was adopted for statistical analysis using GraphPad Prism 8 software. Pearson correlation coefficient was used to analyze the relationship among circ_0001361, miR-525-5p and VMA21. A P value less than 0.05 was set as statistically significant.