Patient tissue samples and cell lines
This study was approved by the ethics committee of the First affiliated hospital of Jinan University. All patients provided a written informed consent. Fresh-frozen cancer tissues and adjacent normal tissues from 30 patients with thyroid cancer were used for this study. Fresh-frozen tissues from 50 patients with benign thyroid nodules were used as control.
Thyroid cancer cell lines (K1, BHT-101, KHM-5M, B-CPAP) were all purchased from the Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were preserved as per the manufacturer’s instructions. Briefly, the cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM penicillin and streptomycin (Gibco BRL, NY, USA). All cell lines were cultured in humidified air supplemented with 5% CO2 at 37 °C.
CircRNAs expression profile analysis
The gene expression profiles (which one) of TC were reviewed from the Gene Expression Omnibus database (GEO, http://www.ncbi. nlm.nih.gov/geo). The online analysis tool GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) was utilized to assess the differentially expressed genes (DEGs) in this study. The adjusted P values were then calculated using the Benjamini and Hochberg false discovery rate method to correct for the occurrence of false positive results.
RNase R resistance analysis of circRNAs
Hsa-circ-0012417 from BHT-101 and B-CPAP was treated with RNase ((4 U/mg, Epicenter) and incubated for 30 minutes at 37 °C. The treated RNAs were then reverse transcribed using specific primers. Next, quantitative real-time PCR (qRT-PCR) was used to determine the expression of has-circ-0012417 in the TC cell lines.
RNA extraction and quantitative real-time PCR (RT-PCR) assay
Total RNA was extracted from the thyroid cancer tissues/cell lines and adjacent normal tissues/cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After assuring RNA quantification and quality using NanoDro2000c (Thermo Scientific, Waltham, USA), 2 µg of total RNA were reverse transcribed to cDNA using the BestarTM qPCR RT kit (#2220, DBI Bioscience, China). Quantitative polymerase chain reaction (RT-PCR) was then done using the BestarTM qPCR MasterMix (#2043, DBI Bioscience,China).
Lentivirus constructs and cell transfection
For in vivo studies, circRNA0012417- knockdown cells (si-circRNA0012417) and negative control cells (NC) were utilized. Lentivirus particles were bought from Hanbio Biotechnology (Shanghai, China). 48 hours after transfection, stable-transfection cells were established by puromycin selection (2 µg/ml) applied for 10 days. LV003-has-circ-0012417 overexpression and negative cells (LV003 vector control) were established in the same way.
The miR-29c, and the miR-miR-29c inhibitor were acquired from GenePharma (Shanghai, China). B-CPAP and BHT101 cells were seeded in 6-well plates and then transfected the next day using Lipofectamine3000 (Invitrogen, CA, USA) as per the manufacturer’s instructions. Cells were harvested at 48 hours post transfection followed by confirmation of transfection efficiency using qRT-PCR.
Cell proliferation assay
The cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8, Dojindo, Osaka, Japan). B-CPAP and BHT101 cells in different groups were seeded into 96-well plates with serum-free DMEM medium. Each group was duplicated with three independent wells. Cell proliferation was observed at 24, 48 and 72 hours. Prior to observation, each well was treated with 10 µl CCK-8 reagent and then cells were incubated at 37℃ for another 3 hours before measuring absorbance at 450 nm utilizing a spectrophotometer. All experiments were done in triplicate.
Transwell migration assay and invasion assay
B-CPAP and BHT101 cells (5 × 104) in different groups were resuspended in 200 µl serum-free medium and seeded into the upper chamber of 24-well plates (Corning, New York, NY, USA) with (invasion) or without (migration) Matrigel (BD Biosciences, New York, NY, USA), while 600 µl medium containing 20% FBS were supplied at the lower chamber as chemoattractant. After incubation at 5% CO2 /37 °C for 20 hours, cells were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 30 min. The number of cells that migrated or invaded was determined in five randomly selected fields using an inverted microscope.
Cell cycle assay
Cells (2 × 106) were collected and fixed with 70% ethanol at 4 °C overnight, and 100 µl RNase A (Keygen Biotech, Nanjing, China) were then added, followed by incubation at 37 °C for 30 min. Cells were then stained with 400 µl propidium iodide (Keygen Biotech) for 30 min. A FACS Calibur flow cytometer (Becton Dickinson) was applied to evaluate cells at 488 nm, and ModFit LT software (Verity Software House) for analysis. All assays were repeated at least three times.
Luciferase reporter assay
A mutant circRNA12417 without miR-29c binding sites were obtained by overlap extension PCR with mutant primers. Briefly, BHT-101 and B-CPAP cells were co-transfected with psiCHECK-circRNA12417-WT or psiCHECK-circRNA12417-Mut and miR-29c mimics or miR-NC using Lipofectamine3000 (Invitrogen). Luciferase activity was measured at 48 hours after transfection by a Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) as per the manufacturer’s instructions.
Total protein was extracted from cells using RIPA lysis buffer (Keygen Biotech). The lysate protein was separated by 10% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk in TBST for 2 h, proteins were then incubated with primary and secondary antibodies. Signal detection was visualized by using enhanced chemiluminescence. Finally, relative concentration was calculated using Quantity One software (Bio-Rad). Primary antibodies included those against CBX8 (dilution 1:1000, 14696) from Cell Signaling Technology, (CA, USA), and Cyclin D1 (dilution 1:200, 60186-1-Ig), Bax (dilution 1:2000, 50599-2-Ig), and GAPDH (dilution 1:5000, 10494-1-AP) from Proteintech (Chicago, USA).
Tumor formation in nude mice
The validated thyroid cancer cell line hsa-circ-0012417 (B-CPAP Circ) and its negative control strain (B-CPAP NC) were expanded and cultured. 1 × 107 cells was injected subcutaneously into 10 BALB/c-nu/nu female nude mice weighing between 18–22 g (4–5 weeks old). 14 days after transfection, the length and width of the tumor were measured once a week for 5 weeks. After 5 weeks, the mice were sacrificed by cervical dislocation. The tumor length, width and weight were all measured and photographed.
Statistical analysis was done using SPSS 18.0 software (SPSS, Chicago). T test was used to analyze the relationship between the indicators of each group. P value less than 0.05 was deemed as statistically significant.