Construction of animal model. Chinese hamsters (n=90) were individually housed in cages with standard laboratory conditions at 25°C, 45% humidity, controlled environment with a 12-hour light/dark cycles. They were randomly divided into three groups: control group (n=30, not any treated), negative control group (n=12, coated with acetone) and model group (n=48, coated with acetone-dissolved 9, 10-Dimethy1-1, 2-Benzanthracence (DMBA)) . After applying, the Chinese hamsters were fasted for 2 hours after the application, and the rest was free to eat and drink. Anatomical samples were performed regularly at weeks 6, 9, 12, 15, 18 and 21: the Chinese hamsters were anesthetized with 0.3% pentobarbital sodium solution by intraperitoneal injection, the left and right cheek pouches of the Chinese hamster were taken, which 1/2 buccal sac tissues were marked and placed in liquid nitrogen for freezed immediately, and then stored at -80°C in the refrigerator; 1/4 buccal sac tissues were fixed in 4% paraformaldehyde for 24 hours, then subjected to alcohol gradient dehydration, paraffin embedding; 1/4 buccal sac tissues were quickly placed in a glutaraldehyde fixative solution and fixed at 4°C. Animal experiments were performed at barrier animal laboratory facility of the Center for Experimental Animal of Shanxi Medical University [SCXK (Jin) 2017-0001] and carried out strictly in accordance with the operating rules formulated by the Institutional Animal Care and Use Committee of Shanxi Medical University (IACUC 2017-016). According to the 3R principle (Reduction, Replacement and Refinement) used by the experimental animals, humane care is given.
HE staining to detect pathologic structure. The paraffin-embedded tissues were subjected to alcohol gradient and xylene dewaxing treatment, 4 𝜇m longitudinal sections were stained with hematoxylin solution for 3 minutes, then rinsed in distilled water twice. Immerse the slice in the 1% acid ethanol for 1 minute and rinse with distilled water. Then the sections were stained with eosin solution for 2 minutes and followed by rinsing in distilled water and dehydration with graded alcohol and clearing in xylene. Finally, the pathological results were observed under a fluorescence microscope (IX70, Olympus).
Ultrastructural observation by Scanning Transmission Electron Microscopy (STEM). The buccal sac tissue fixed by glutaraldehyde was immersed in phosphate buffer solution, then immobilized in osmium tetroxide solution, dehydrated by ethanol gradient, and embedded in epoxy resin No. 812. After polymerization, the buccal sac tissue was sliced by ultrathin slice machine, stained with uranyl acetate and lead citrate, and observed by transmission electron microscopy under a microscope.
RNA extraction and quality test. Total RNAs were extracted from frozen tissues and OSCC cells using miRNeasy Mini Kit (External content QIAzol Lysis Reagent), according to the manufacturer’s instructions. Then the RNA qualities were evaluated by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). The RNA purity is qualified by the OD260/OD280 ratio, which ranges from 1.8 to 2.0, and RNA integrity was assessed by 1% agarose gel electrophoresis analysis. Total miRNA was extracted from the OSCC cells using miRNA extraction and purification kits, preserved at -80℃ before using.
Library construction and sequencing. After RNA extraction and quality test, a quantity of 1 μg total RNA of tissues was constructed library, using TruSeq Small RNA Library Prep Kit for Illumina following the manufacturer’s protocol. Removed ribosomes RNAs, enriched circRNAs were short fragment by using fragmentation buffer. First strand cDNA was synthesized using reverse transcriptase with random hexamer primers. Second strand cDNA was synthesized by DNA polymerase I, dNTP, RNase H. Next, the cDNA fragments were purified with AMPure XP beads. The purified double-stranded cDNA was repaired end, poly(A) added and ligated to Illumina sequencing connector, then, AMPure XP beads were used to select the fragment size, the target fragment was recovered by agarose gel electrophoresis. The obtained fragment was subjected to PCR amplification and enrichment, the cDNA library was constructed. The constructed cDNA library was quantified using Qubit2.0. Which was tested by Agilent 2100 Bioanalyzer and ABI StepOne plus Real-time PCR System. After passing the quality inspection, the effective concentration of the library (more than 2nM) was quantified accurately by qPCR to ensure the quality of the library. Sequenced using Illumina Hiseq2500 SR50. Each sample was blasted to the genome by quality-controlled PE clean reads data and the Chinese hamster reference genome (GCF_000223135.1_GriGri_1.0_genomic.fa, NCBI). The comparison software uses the BWA-MEM algorithm required by CIRI, a circular RNA identification tool.
CircRNA expression standardization and data analysis. All raw reads were obtained from the sequencing machines. The absolute expression of circular RNA is generally measured by detecting the number of sequences aligned to the head-to-tail locus, but since the total amount of sequencing of each sample library is different, so standardization is required. The RPM (read per million) method commonly used in data analysis was used to normalize the expression level of circular RNA. According to the screening threshold of RPM>0.1, a total of 3485 circRNAs were obtained that satisfied the expression requirements. Differential expression analysis of model and control groups was performed using edgeR package. We identified circRNAs with log2 |(Fold change)|>2 and P-value <0.05 as significant differentially expressed circRNAs, then circRNAs were annotated with Cricetulus griseus genome. Those cannot be annotated were defined as novel circRNAs.
Functional enrichment and pathway analysis. During functional enrichment analysis of differentially expressed circRNAs, the online analysis tool-Metascape was utilized to perform gene ontology (GO) enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis associated genes of differentially expressed circRNA.
Integrated analysis of circRNAs-miRNAs in Chinese hamster tissue. The target relationship of annotated circRNAs with miRNAs were theoretically predicted by conserved seed-matching sequence using miRanda (v3.3a) analysis, which the maximum binding energy required is less than -20, and there are at least two miRNA binding sites on a circular RNA sequence. The analysis showed all circRNAs contained their respective miRNA response elements (MREs) and the circRNA-miRNA network was visualized by Cytoscape 3.01.
Cells culture. The human cell lines CAL27 and Tca8113 were purchased from Procell and Boster Biological Technology (Wuhan, China). OSCC cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were cultured at 37℃ with 5% CO2.
RNA interference and transient cell transfection. The small interfering RNA (siRNA) and negative control siRNA (si-NC) were synthesized by GenePharma (Shanghai, China) to inhibit the expression of hsa_circ_0127523 in CAL27 and Tca8113 cells. Si-hsa_circ_0127523 siRNA (si-hsa_circ_0127523-1: sense: 5ʹ-ACAUACAGGGAGUGAAACCTT-3ʹ; antisense: 5ʹ-GGUUUCACUCCCUGUAUGUTT-3ʹ; si-hsa_circ_0127523-2: sense: 5ʹ-UAUACCACAUACAGGGAGUTT-3ʹ; antisense:5ʹ- ACUCCCUGUAUGUGGUAUATT-3ʹ) and negative control siRNA (si-NC:sense: 5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ; antisense: 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ) were transfected into CAL27 and Tca8113 cells using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, USA). After the operation, place the 6-well plate in the incubator as soon as possible. Transfection sequence after inoculation of cells for 24 hours.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Total RNA was reverse transcribed to cDNA by random hexamer primers (Takara, Japan). SYBR green PCR Master Mix (Takara, Japan) was used to examine circRNA, miRNA and mRNA expression, circRNAs and mRNAs using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control, miRNAs using U6 as an endogenous control, every group at least three tissues and repeat at least three times, which monitored by the ABI StepOne Plus Real-time PCR System. The melting curve insure the specificity of primers. Relative circRNAs, miRNAs and mRNAs expression were calculated according to the 2-ΔΔCT method. The primers were designed and synthesized by using Oligo 6.0 software and Shanghai Sangon Biotech (Table 2, 3).
Cell Counting Kit-8 (CCK-8) assay. The proliferation of CAL27 and Tca8113 cells were detected by the CCK-8 assay. Infected cells were plated into 96-well plates at 3×103 cells/well. At 0, 24, 48 and 72 hours, according to the manufacturer’s guidelines, 10 μl of CCK-8 liguid (Boster Biological Technology, Wuhan, China) was added adherence and incubation for 2 hours. The optical density (OD) was detected by a microplate reader at 450 nm.
Transwell migration and matrigel invasion assays. The migration and matrigel invasion assays were performed using 24-well transwell plates, using transwell chamber and pre-coated matrigel transwell chamber respectively. The infected cells were suspension in DMEM without FBS and added to the upper chambers (2×104 cells in 200 μl medium), while the lower chambers were added DMEM including 10% FBS (700 μl) and incubated for 48 hours. Compared with the upper chambers of migration assay, the upper chamber of invasion assay was added with matrigel. After incubation 48 hours, the cells and matrigel in upper chambers were removed, the cells which migration or invasion were fixed 30 minutes in 4% paraformaldehyde and stained 1 hour with 0.1% crystal violet, taken photos and counted.
Online prediction in human cells. CircRNA-miRNA interaction was predicted with the software CircInteratome (https://circinteractome.nia.nih.gov), miRanda (http://www.microrna.org/microrna/microrna/home.do) and Targetscan (http://www.targetscan.org/vert_72).
Statistical analysis. All data were statistical analyses by SPSS 22.0 and GraphPad Prism 8.0 software. Demonstrate significant difference circRNAs from high-throughput sequencing were analyzed by t-test between model and control groups. The results of PCR, CCK-8, migration and invasion were presented as mean ± SEM. Statistically significant data (P<0.05) were determined by Student t-test. The pairwise comparison between the multiple groups of samples was performed by the LSD method in one-way analysis of variance (ANOVA).