Cell culture and treatment
Among three OSCC cell lines (SCC9, SCC15 and SCC25), SCC9 and SCC15 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) while SCC25 cells were from Procell Life Science & Technology Co., Ltd. (Wuhan, China). A healthy human oral keratinocyte (HOK) cell line was purchased from Binsui Biotechnology Co., Ltd. (Shanghai, China). All the cell lines were cultivated in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Gaithersburg, MD, USA) and supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/ml penicillin/streptomycin under 5% CO2 at 37 °C. For circRNA characterization, 3 U/mg of RNase R from Epicentre Technologies (Madison, WI) and 2 mg/ml of Actinomycin D (Act D) from Sigma-Aldrich (St. Louis, MO) were used.
Cell transfection
To knock down circZDBF2 or RNF145 expression, short hairpin RNAs (shRNAs) specifically targeting circZDBF2 (sh-circZDBF2-1/2/3) or RNF145 (sh-RNF145-1/2/3), together with the negative control (NC) were synthesized from GenePharma (Shanghai, China). In addition, pcDNA3.1/circZDBF2, pcDNA3.1/RNF145 and pcDNA3.1/CEBPB, along with their negative control pcDNA3.1 obtained from GenePharma were constructed respectively for the overexpression of genes. The miR-362-5p mimics/inhibitor, miR-500b-5p mimics/inhibitor and their negative controls (NCs) (mimics-NC/inhibitor-NC) were designed via Ribobio (Guangzhou, China). Cell transfection was taken using Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA). Forty-eight hours later, cells were collected for further investigations.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted in SCC9 and SCC15 cells by the application of TRIzol® reagent (Takara, Japan) in line with the protocols of supplier. Prime Script™ RT Master Mixture (Takara 11141ES10, Japan) or TaqMan® MicroRNA RT kit (4366596, Applied Biosystems™, Foster City, CA, USA) was utilized for RNA reverse transcription (RT). Then, qPCR was implemented with the qRT-PCR Kit (QR0100-1KT, Sigma-Aldrich, USA) by using StepOnePlus™ Real-time PCR Systems (Applied Biosystems™). Relative expression levels were calculated using the 2−ΔΔCt method, with U6 small nuclear RNA (U6) as the endogenous control to normalize miRNA expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control to normalize mRNA/circRNA expression. Related primer sequences were exhibited in Additional file 8: Table S1.
Cell counting kit-8 (CCK-8) assay
Transfected SCC9 and SCC15 cells harvested at the logarithmic growth phase were incubated into 96-well plates (5 × 103 cells/well) with complete culture medium at 37 ℃ for 24, 48, 72 h. Then, cells were cultivated with 10 μL of CCK-8 reagent (ab228554, Abcam, UK) for 1 h. The absorbance at 450 nm was assayed by microplate reader.
Colony formation assay
Transfected SCC9 and SCC15 cells were prepared in 6-well plates (800 cell/well) for 14 days of colony formation. The culture medium was discarded and the cells were washed with phosphate buffer saline (PBS) for two times. The colonies were fixed with methanol and dyed by 0.5% crystal violet solution. Finally, colonies with no less than 50 cells were counted manually.
Transwell-invasion assays
Serum-free medium containing transfected SCC9 and SCC15 cells was planted on the top of 24-well transwell chambers (5 × 103 cells/well) along with Matrigel, and the lower chambers were loaded with complete medium. Twenty-four hours later, the medium was discarded and non-invaded cells were removed by a cotton swab. The bottom of the chamber was fixed by methanol solution for 15 min and dyed with crystal violet for 10 min. The cells that had invaded were counted at 5 randomly chosen visual fields under a microscope (Smart zoom 5, Zeiss).
Wound healing assay
Cell samples were seeded in 6-well plates (1 × 106 cells/well) for reaching 100% cell confluence, and then treated with 200-μL pipette tip. The scratched cells were removed, the serum-free medium was added, and then the cells were cultivated in an incubator at 37 °C with 5% CO2. The width of 3–5 randomly selected spots at 24 h was recorded and the distance of wound closure was analyzed.
Western blot
Total protein extracted from SCC9 and SCC15 cells was isolated by Radio Immunoprecipitation Assay (RIPA) lysis buffer and then subjected to isolation through electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE;10%). The samples transferring on polyvinylidene fluoride (PVDF) membranes were blocked via 5% nonfat milk and then subjected to incubation with primary antibodies against ZO-1 (ab276131, Abcam, UK), E-cadherin (ab40772, Abcam, UK), N-cadherin (ab76011, Abcam, UK), Vimentin (ab92547, Abcam, UK), p50 (ab32360, Abcam, UK), p65 (ab16502, Abcam, UK), IκBɑ (#9242, Cell Signaling Technology, China), and GAPDH (China Kangcheng Biotechnology Co., Ltd.) as the internal control. After being rinsed in Tris-buffered saline-tween (TBST), samples were incubated with secondary antibody labeled with horseradish peroxidase (HRP) and finally exposed to electrochemiluminescent (ECL) luminescent liquid (Santa Cruz Biotechnology, Santa Cruz).
Fluorescence in situ hybridization (FISH) assay
The circZDBF2-specific RNA FISH probe procured from Ribobio was used for cellular analysis as instructed by provider. The fixed cell samples were rinsed in PBS, and then air-dried, followed by the hybridization with FISH probe. Cell nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) solution purchased from Beyotime (Shanghai, China), and the fluorescent images were captured by GLomax20/20 fluorescence microscope (Promega).
RNA immunoprecipitation (RIP) analysis
RIP analysis was made via Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) based on user guides. SCC9 and SCC15 cells were lysed by RIP buffer and cell lysates were collected. Magnetic beads were conjugated with argonaute2 antibody (anti-AGO2; ab186733, Abcam, UK) or immunoglobulin G antibody (anti-IgG; ab205718, Abcam, UK). After rinsing, the precipitated RNA went through qRT-PCR analysis.
Chromatin immunoprecipitation (ChIP) assay
ChIP was conducted utilizing EZ-ChIP chromatin immunoprecipitation kit (Millipore, Bedford, USA) in accordance with user guides. SCC9 and SCC15 cells were cross-linked in 4% paraformaldehyde, sonicated into chromatin fragments of 200–1000-bp and incubated with the antibodies against CEBPB (ab264305, Abcam, UK), with Anti-IgG served as the negative control. The specific primers for the RNF145 promoter were used, and precipitated DNA was detected by qRT-PCR.
RNA pull down assay
Based on the protocols of supplier, we utilized the Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA) to perform this assay. Biotin-labeled circZDBF2 (Bio-circZDBF2) or RNF145 (Bio-RNF1452) probes were incubated with cell extracts and streptavidin magnetic beads, with Bio-NC as the negative control. Finally, the RNA complexes bound to beads were analyzed by qRT-PCR. In this study, three experimental groups (Bio-NC, Bio-circZDBF2/RNF145-WT and Bio-circZDBF2/RNF145-Mut) were constructed respectively to evaluate the binding capacity between circZDBF2/RNF145 and miR-362-5p/miR-500b-5p.
Dual-luciferase reporter analyses
The wild-type (WT) and mutant (Mut) miR-362-5p or miR-500b-5p binding sites within circZDBF2 sequence or RNF145 3′UTR (3′ untranslated region) were sub-cloned into pmirGLO dual-luciferase vector, and pmirGLO-circZDBF2-WT/Mut or pmirGLO-RNF145 3’UTR-WT-Mut plasmids were hence constructed. The pmirGLO plasmids were co-transfected with miR-362-5p mimics or miR-500b-5p mimics and their NC mimics in SCC9 and SCC15 cells. Forty-eight hours later, the luciferase activity was estimated with Dual-Luciferase Reporter Assay System (Promega Corporation, Fitchburg, WI).
In vivo tumor growth assay
Total of 2 × 106 SCC9 cells transfected with sh-NC or sh-circZDBF2-1 were injected into the male BALB/c nude mice (6–8-week old; total number = 8 mice; n = 4 mice/group; Slac Laboratories, Shanghai, China). The animal assay was performed strictly in line with the protocol approved by the Ethical Committee of Affiliated Jinling Hospital, Medical School of Nanjing University, with the approval numbered 2020DZGZRZX-078. Tumor volume was calculated every 3 days following the formula: volume = length × width2/2. Four weeks later, mice were sacrificed after which tumors were excised and weighed.
Immunohistochemistry (IHC)
Paraffin-embedded tissues from in vivo tumor growth assay were first fixed by 4% paraformaldehyde, embedded in paraffin and cut into consecutive sections at 4-μm thick for IHC assay. Then sections were incubated with anti-Ki67 (ab15580, Abcam, UK) at 4 °C overnight, and then with secondary antibody for 30 min. Images were taken using an Olympus microscope (Olympus Corporation, Japan).
Statistical analysis
Each experiment was performed for three times in this research. Statistical analysis was made via SPSS 23.0. Mean ± standard deviation (SD) was used to show the results. After validating normal distribution via shapiro test and homogeneity of variance via Levene test, data were analyzed via Student’s t-test or one-way analysis of variance (ANOVA) for difference analyses. P < 0.05 was of great importance for statistically significant difference.