Clinical sample collection
HCC patients and healthy volunteers were recruited from Henan Province Hospital of Traditional Chinese Medicine. HCC tissues (n=32) and matched surrounding normal tissues (n=32) were obtained from HCC patients, and HCC serum specimens (n=32) and normal serum specimens (n=26) were obtained from HCC patients and healthy volunteers, respectively. These fresh tissues and serums were frozen and stored at -80℃ conditions until further use. Prior patient’s informed consent was signed, and this research acquired the approval from the Institute Research Ethics Committee of Henan Province Hospital of Traditional Chinese Medicine.
Cell lines
Two HCC cell lines, including SNU-387 and Huh7, and immortalized human hepatocytes (THLE-2) were purchased from YaJi Biological (Shanghai, China). SNU-387 cells were cultured in Roswell Park Memorial Institute (RPMI-1640; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS). Huh7 cells were kept in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% FBS.
THLE-2 cells were maintained in Bronchial Epithelial Cell Growth Medium (BEGM; Gibco) containing 10% FBS. All cells were maintained in a thermostatic incubator at a constant temperature of 37℃ with moist air and 5% CO2.
Circular structure analysis
The sequence of circ_0015756 was obtained from circbank (http://www.circbank.cn/), and then the sequence was further analyzed by the Genome Browser tool (https://genome.ucsc.edu/), including back-splicing site and exon regions.
Cell transfection
We used small interference RNA (siRNA) to decrease circ_0015756 expression. SiRNA targeting circ_0015756 (si-circ_0015756) plus its negative control (si-NC), miR-610 mimic plus its negative control (miRNA NC) and miR-610 inhibitor plus its negative control (inhibitor NC) were all synthesized by Ribobio Co. Ltd (Guangzhou, China). For FGFR1 overexpression, FGFR1 sequence was cloned into pcDNA overexpression vector, generating FGFR1 overexpression vector (pc-FGFR1), and empty pcDNA was served as negative control (pc-NC). Construction of expression vector was accomplished by Sangon Biotech (Shanghai, China). These transfection contents were introduced into SNU-387 and Huh7 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using TRIzol reagent (Qiagen, Duesseldorf, Germany). For circRNA and mRNA analysis, PrimeScript RT Master Mix (Takara, Tokyo, Japan) and TB Green Premix Ex Taq (Takara) were used for reverse transcription and qRT-PCR, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. For miRNA analysis, miScript reverse transcription kit (Qiagen), and miScript SYBR Green PCR kit (Qiagen) were utilized, with U6 as the housekeeping gene. The relative expression was calculated using the 2−ΔΔCt method, and all used primers were listed as below: circ_0015756, F: 5’-AATGGATGGAGCCAGTAA-3’ and R: 5’- AAACCACCCTCACAAGTA-3’; CFH, F: 5’-CAGCAGTACCATGCCTCAGA-3’ and R: 5’-GGATGCATCTGGGAGTAGGA-3’; miR-610, F: 5’- AGAAGGCTGGGGCTCATTTG-3’ and R: 5’-AGGGGCCATCCACAGTCTTC-3’; FGFR1, F: 5’-CCTGGTGACAGAGGACAATG-3’ and R: 5’- AGATCCGGTCAAATAATGCC-3’; GAPDH, F: 5’-GACTCAT GACCACAGTCCATGC-3’ and R: 5’-AGAGGCAGGGATGATGTTCTG-3’; U6, F: 5’-CTCGCTTCGGCAGCACA-3’ and R: 5’-AACGCTTCACGA ATTTGCGT-3’.
Cell counting kit-8 (CCK-8) assay
CCK-8 assay kit (Beyotime, Shanghai, China) was used to examine cell viability. Briefly, 2 × 103 transfected cells were seeded into 96-well plates. After 24 h post-transfection, 10 μL CCK-8 reagent was pipetted into every well, reacting for another 2 h. The absorbance at 450 nm was measured by the microplate reader (BioTek, Winooski, VT, USA).
Colony formation assay
Cells with transfection were incubated for 24 h, and then cells were digested with trypsin and replanted into 10 cm2 plate. After culturing for 2 weeks, cells were subjected to methanol and 0.1% crystal violet. Colonies larger than 100 µm in diameter were counted under a microscope (Olympus, Tokyo, Japan).
Transwell assay
SNU-387 and Huh7 cells (3 × 104) with transfection in serum-free culture medium were transferred into the upper chambers (BD Biosciences, San Jose, CA, USA) for migration analysis or chambers pre-treated with Matrigel (BD Biosciences) for invasion analysis. Meanwhile, the bottom of chambers was padded with corresponding fresh culture medium (containing 10% FBS). Over the next 24 h, cells in 37℃ conditions were allowed to migrate and invade. The migrated or invaded cells in the lower surface were fixed with methanol and dyed with 0.1% crystal violet, followed by observation under a microscope (100 × magnification; Olympus).
Cell cycle distribution and cell apoptosis analyses
SNU-387 and Huh7 cells with transfection were seeded into 6-well plates (2 × 105 cells/well). After culturing for 24 h, cell cycle was detected using cell cycle analysis kit (Vybrant DyeCycle; Invitrogen) in line with the manufacturer’s guideline and analyzed by flow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
SNU-387 and Huh7 cells with transfection for 48 h were collected and washed with phosphatic buffer saline (PBS). Then, cells (6 × 104) were used to conduced apoptosis analysis using the Annexin V-FITC and PI Apoptosis Detection Kit (FITC: fluorescein isothiocyanate; PI: propidium iodide) (Beyotime) following the protocol. The apoptotic cells were analyzed by flow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
In vivo experiments
For circ_0015756 stable downregulation, short hairpin RNA lentiviral vector containing circ_0015756 (sh-circ_0015756) and its negative control (sh-NC) were assembled by Sangon Biotech. In Xenograft models, Huh7 cells transfected with sh-circ_0015756 or sh-NC were implanted into the right side of the nude mice (4~6 weeks-old, BALB/c, n=10) purchased from Charles River (Beijing, China). All the nude mice were housed in the specific pathogen-free animal room. After housing for one week, tumor volume was recorded once a week, and tumor weight was measured after 28 d when mice were killed. All animal experiments were carried out with the approval of the Animal Care and Use Committee of Henan Province Hospital of Traditional Chinese Medicine .
Target gene prediction
The online bioinformatics tool circRNA interactome (https://circinteractome.nia.nih.gov/) was used to predict the targets of circ_0015756, and another bioinformatics tool Targetscan (http://www.targetscan.org/vert_72/) was used to predict the targets of miR-610.
RNA pull-down assay
The sequence of mutations in miR-610 at the binding site with circ_0015756 was designed, named as MUT-miR-610. Then, miR-610, miRNA NC and MUT-miR-610 were subjected to biotinylation in Ribobio Co. Ltd., terming as biotin-miR-610 mimic, biotin-miRNA NC and biotin-miR-610 mimic MUT. Then, all of them were individually transfected into SNU-387 and Huh7 cells. After 48 h, transfected cells were lysed, and cell lysates were incubated with Dynabeads MyOne Streptavidin beads (Invitrogen). Subsequently, RNA was extracted using TRIzol Reagent (Qiagen) from the beads and detected by qRT-PCR.
RNA immunoprecipitation (RIP) assay
SNU-387 and Huh7 cells were transfected with miR-610 mimic, miR-610 mimic MUT or miRNA NC. Transfected cells were then lysed, and cell lysates were subjected to RIP buffer mixed with magnetic beads coated with Argonaute 2 antibody (anti-Ago2) or Immunoglobulin G antibody (anti-IgG; control). After sufficient mixing and washing, the complexes on beads were analyzed using qRT-PCR.
Dual-luciferase reporter assay
The sequence fragment of circ_0015756 containing miR-610 binding site and the corresponding mutant sequence fragment were synthesized and inserted into the pmirGLO expression vector (Promega, Madison, WI, USA), namely WT-circ_0015756 and MUT-circ_0015756. Similarly, WT-FGFR1 3’UTR and MUT-FGFR1 3’UTR were also generated following the same manner. Subsequently, SNU-387 and Huh7 cells were cotransfected with miR-610 mimic and WT-circ_0015756, MUT-circ_0015756, WT-FGFR1 3’UTR or MUT-FGFR1 3’UTR, and miRNA NC and these fusion plasmids were also cotransfected into SNU-387 and Huh7 cells as the control. After 48 h-transfection, the luciferase activity of cells was measured using the Dual-Luciferase Assay System (Promega).
Western blot
Protein samples were prepared and detected using the BCA protein assay kit (Beyotime). Then, equal amount (20 μg) of protein was used in the Western blot analysis. The primary antibodies used were anti-FGFR1 (ab76464; Abcam, Cambridge, MA. USA) and GAPDH (ab9485; Abcam). The secondary antibody used was anti-Goat anti-Rabbit (ab205718; Abcam). The protein blots were emerged using an enhanced chemiluminescence (ECL) kit (Beyotime).
Statistical analysis
All data were collected from 3 groups of independent experiments and showed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 5 software (San Diego, CA, USA). Comparisons between two groups were executed using Student’s t tests, and comparisons among three or more groups were conducted using analysis of variance with Tukey post-hoc test. Statistical differences were defined as P < 0.05.