Human NSCLC tissues
A total number of 50 NSCLC tumor samples and matched non-carcinoma lung samples were collected at the Department of Lung Cancer, Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). This study was approved by the medical research ethics committee of Tianjin Medical University Cancer Institute and Hospital. All the patients signed the informed consent before surgery. The NSCLC tumor samples and adjacent normal samples were collected and snap-frozen in liquid nitrogen freezing before the preservation under − 80 °C.
Cell culture and transfection
Human bronchial epithelial cells (HBE) and human NSCLC cells (CALU3, CALU6, A549, H1229), and human renal epithelial cell line (H293T) were acquired from American Type Culture Collection (Virginia, USA). Cells were cultured win DMEM that contained 10% FBS (Gibco, NY, USA) and 100 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma, Germany) in a humid incubator under 5% CO2 at 37 °C. pcDNA3.1-FZD4 expression plasmid, small interfering RNA (siRNA) of circ_0017109, control siRNA, miR-671-5p inhibitor, miR-671-5p mimics and the corresponding negative controls (NCs) were provided by Genomeditech Co., Ltd. (Shanghai, China). Transfections were performed with Lipofectamine 2000 (Invitrogen) in line with specific instructions. Briefly, in 6-well plates, 60% confluent cells were transfected with 6 μg of pcDNA3.1-FZD4, or 50 nM of miRNA mimic, inhibitor or siRNA according to manufacturer’s instruction. After transfection for 48-h, the cells were collected for further experimental analysis.
Quantitative real-time PCR (qRT-PCR)
TRIzol reagent (Invitrogen, USA) was employed for RNA extraction. 1 μg of RNA sample was prepared into cDNA using PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan) in line with specific protocols. CFX96 Touch Real-time PCR detection system (Bio-Rad, Hercules) was adopted for qPCR analysis using specific primers and SYBR Premix Ex Taq II (Takara). GAPDH or U6 was used as controls for protein-coding gene or non-coding RNA respectively, and the results were analyzed using the 2−ΔΔCT method. For miR-671-5p, reverse transcription was performed using Taqman MiRNA Reverse Transcription Kit (Applied Biosystems, MIMAT00038800). The primer sequences used in the study were as follows: circ_0017109, 5′-AGATTCCGTTCGGCTCCTCC-3′ (F) and GCACTGGTGTCTGTTG TTAGC-3′ (R); FZD4, 5′-CCTCGGCTACAACGTGACC-3′ (F) and 5′-TGCACATT GGCACATAAACAGA-3′ (R); U6, 5′-GACAGTCAGCCGCATCTTCT-3′ (F) and 5′-GCGCCCAATACGACCAAATC-3′ (R); GAPDH; 5′-CTCGCTTCGGCAGCACA-3′ (F) and 5′-AACGCTTCACGAATTTGCGT-3′ (R).
Treatment with RNase R
A total amount of 5 μg RNA was equally divided into two portions: one was treated with 3 units of RNase R (Epicenter, Madison, WI) under 37 °C for 15 min, and the other was incubated at the same condition without RNase R (Mock). After treatment, RNA samples were collected by RNeasy MinElute Cleaning Kit (Qiagen, Germany), which was further analyzed via qRT-PCR.
Subcellular fractioning
Nuclear and Cytoplasmic RNA Purification Kit (Norgenbiotek Corporation, Thorold, Canada) was adopted for the nuclear and cytoplasmic fractioning, and the subsequent RNA isolation from A549 and H1299 cells. Circ_0017109 expression levels in different fractions were evaluated through qRT-PCR, with U6 and GAPDH being nuclear and cytoplasmic references, respectively.
Colony formation and CCK-8 proliferation assay
For colony formation experiment, cells (1000/well) were seeded into the 6-cm culture dish, and cultured for 2 weeks with medium replenishment every 2 days. On day 14, cells were subjected to 4% paraformaldehyde (PFA) fixation and the staining of 0.4% crystal violet (Sigma, Germany) for 20 min. Colonies that contained over 10 cells were counted under light mode of an FV1000 microscope (Olympus, Tokyo, Japan), at 200X magnification.
CCK-8 kit (Abcam, ab228554) was utilized to detect cell proliferation following specific instructions. 48 h post transfection, 2000 cells were seeded into each well in 96-well plates and the cells were cultured for different durations. At indicated time point, the cells were mixed with CCK-8 reagent (20 μL/well), followed by 2-h incubation under 37 °C. The absorbance (OD) measurement was conducted using a microplate reader at 450 nm.
Flow cytometry analysis of apoptosis
For quantifying apoptosis level, Annexin V-FITC/Propidium Iodide kit (BCT- Adipogen，XAP2102-TT01) was used to stain cells following the manufacturer’s instructions. Briefly, 1 × 106 cells in 1 ml Annexin V staining buffer was mixed with 1 μl Annexin V dye and 1 μl Propidium Iodide reagent. After 15-min staining, the cells were washed twice with staining buffer and then analyzed using BD FACSCalibur™ Flow Cytometer (BD Biosciences).
Western blotting (WB) assay
Protein samples collection was performed using RIPA buffer (KeyGen, Shanghia, China) on ice for 15 min. Total protein contents were quantified by an Enhanced BCA Protein Assay Kit (Beyotime, Beijing, China). Aliquots of protein were loaded onto 10% SDS-PAGE gel, followed by transfer onto PVDF membranes (Millipore, Darmstadt, Germany). The membranes were subjected to blocking using 5% bovine serum albumin (BSA) for 1 h. The incubation with primary antibodies was performed at 4 °C overnight: cleaved caspase-3 (1:2000, Abcam, ab2302), Bcl-2 (1:2000; Abcam, ab59348), FZD4 (1:1500; Bioss, bs-13217R), β-catenin (1:1500; Bioss, bs-1165R), non-phospho (Active) β-catenin (1:1000; Cell Signaling Technologies, 8814), cyclin D1 (1:2000; Abcam, ab134175), c-Myc (1:1500; Abcam, ab190026), or GAPDH (1:5000; Bioss, bs-2188R). The next day, the membranes were washed 4 times with TBST buffer and then further labeled with HRP-linked secondary antibody (1:10000; Invitrogen, 61–6520) for 1 h at room temperature. After rinsing for 4 times signal development was conducted using a chemiluminescence kit (Santa Cruz, TX, USA), and the protein bands were photographed under a gel imager (Biorad, CA, USA).
Luciferase reporter assay
H293T cells were used for dual luciferase reporter assay. In 96-well plates, cells were transfected with dual luciferase reporter with wild type (WT binding site or the reporter with mutated binding sequences (MUT), in the presence of miR-671-5p mimics or miR-NC. After 48 h, Luciferase Reporter Assays Substrate Kit (Abcam, ab228546) was adopted for measuring the relative luciferase activities of Firefly and Renilla luciferase in line with specific protocols.
RNA pull-down assay
Cells were transfected with 50 nM of biotinylated negative control (bio-NC) or miR-671-5p mimics (bio-miR-671-5p) using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, USA). 48 h post transfection. Cell lysate was collected using IP lysis buffer (Beyotime, Beijing, China), with 10% lysate being saved as the input sample. The remaining sample was mixed with 50 μl C-1 streptavidin magnetic beads (Invitrogen, USA) under 4 °C for 2 h. The beads were then washed 4 times with IP lysis buffer, followed by RNA sample purification with RNeasy Mini Kit (Qiagen, Germany). The relative level of circ_0017109 in each sample was determined by qRT-PCR.
RNA immunoprecipitation
Cells were lysed using an Imprint® RNA Immunoprecipitation Kit in line with specific instructions (Sigma, Germany). One milliliter of cell lysate (1 million cells) was mixed with 5 μg anti-Ago2 or 5 μg IgG isotype, which was immobilized on protein-A magnetic beads. The mixture was incubated at 4 °C overnight. The beads were rinsed 3 times using the immunoprecipitation buffer, and the RNA samples associated with the beads were extracted using TRIzol reagent (Invitrogen, USA). qRT-PCR was employed for measuring the immunoprecipitated RNAs.
Mouse xenograft assay
A549 cells with stable sh-circ_0017109 expression were generated by lentiviral transduction. pLKO.1-Puro lentiviral vector with circ_0017109 shRNA or scramble control shRNA (sh-NC) was provided by Genomeditech Co., Ltd. (Shanghai, China). The production of lentivirus was conducted in 293 T cells by GenePharma Co. Ltd. (Shanghai, China). Cells infected with lentivirus were selected with 800 ng/mL puromycin for 2 weeks before inoculation. Adult BALB/c nude mice (~ 25–30 g) were provided by National Resource Center for Mutant Mice of China (Nanjing, China). Nude mice were raised in a controlled environment at 22 °C with 12 h light/dark cycle. The animals were assigned to two groups (n = 6 each). To establish mouse xenograft model of NSCLC, each mouse was injected with 2 × 106 A549 cells (stable expression of circ_0017109 shRNA or sh-NC) in PBS (150 μL) subcutaneously via flanks. Tumor size was measured every week using a caliber. After 4 weeks, euthanasia of mice was performed using 20% of carbon dioxide in a closed chamber for 15 min until no movement was observed. Tumor was then dissected for subsequent analysis. All the animal experimental protocols were conducted in compliance with animal use and care guidelines of Tianjin Medical University Cancer Institute and Hospital, and were approved by the corresponding animal experimental committee.
Statistical analysis
SPSS19.0 and Prism (GraphPad Sofeware 7.0) were adopted for statistical analysis. Data were displayed as mean ± SD. Student’s t-test (two-tailed) was adopted for two group comparison, and one-way or two-way ANOVA was utilized for multiple comparisons. Pearson’s correlation coefficient was utilized to analyze associations of gene expressions. Kaplan Meier curve was plotted for assessing the overall survival of NSCLC patients, while results were examined using log-rank t-test. The different was considered as significant if p < 0.05.
