Clinical tissue samples
All noncancerous nasopharyngeal epithelial (NPE) tissues and NPC tissues were collected from the Second Xiangya Hospital of Central South University (Changsha, China). All tissues for experimental use were collected with the authorization of the Ethics Committee of Central South University and written informed consents were obtained from the patients. The clinicopathological features of the NPC patients are shown in Table 1. All biopsy specimens were confirmed by the Pathology Department and were immediately frozen in liquid nitrogen for future use.
Cell lines and cell culture
The human normal NPE cell line NP69 and NPC cell lines CNE1, CNE2, 5-8F, and 6-10B were kindly provided by the Cancer Center of Sun Yet-Sen University (Guangzhou, China) and preserved in our laboratory. The HNE1, HNE2, HONE1 and HK1 cell lines were purchased from the Cell Center of Xiangya School of Medicine at Central South University (Changsha, China). All cells mentioned above were cultured in Dulbecco's Modified Eagle’s medium (DMEM; Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS; BI, Jerusalem, Israel), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and contained 5% CO2 atmosphere.
Cell transfection
To construct a plasmid overexpressing circBRD7, the total length sequence of circBRD7 was inserted into a circular RNA vector pcDNA3.1(+) circRNA Mini plasmid (generously provided to us by Prof. Li Yong at Baylor College of Medicine). Two circBRD7 small interfering RNAs (siRNAs; sicircBRD7#1 and sicircBRD7#2), two BRD7 siRNAs (siBRD7#1 and siBRD7#2), hsa-miR-498 mimic, hsa-miR-944 mimic and negative controls were all purchased from RiboBio (Guangdong, China). The sequences of the circBRD7 siRNAs were as follows: sicircBRD7#1: 5’-AGUUAUCCUGACUGUUCACdTdT-3’; sicircBRD7#2: 5’-UUUGAAGUUAUCCUGACUGdTdT-3’. Moreover, siBRD7#1 and siBRD7#2 were identified as effective siRNAs of BRD7 in our previous study, and the sequences were referred as our previous publication [18]. In this paper, we used pool of this two BRD7 siRNAs to perform recovery experiments. The 5-8F and CNE2 cells were digested with trypsin (BI, Israel), counted, and evenly seeded in 6-well plates. The plasmids were transfected when the cell density reached 60-80%, and the siRNAs were transfected when the cell density reached 30-50%. Plasmids, siRNA sequences and microRNA mimics were transfected into cells using Lipofectamine 3000 Reagent (Invitrogen, USA) according to the manufacturer’s instructions.
RNA isolation, RT‑PCR and quantitative real-time PCR
Total RNA was extracted from cell lines and tissues using AG RNAex Pro Reagent (Accurate Biology, Hunan, China) and subjected to reverse transcription applying the reverse transcription kit (Thermo Scientific, Waltham, USA). Real-time PCR was performed with the ChamQ Universal SYBR qPCR Master Mix qPCR Kit (Vazyme, China), and GAPDH or U6 was used as an endogenous control.The RT-qPCR primers sequences used for detection of BRD7, circBRD7 and other potential circRNAs derived from BRD7 are list in Table 2 and Additional file 1: Table S1, respectively. For miRNA detection, a miDETECT A TrackTM miRNA qRT-PCR Starter kit (RiboBio, Guangzhou, China) was used for reverse transcription and RT-qPCR, and U6 was used as the internal reference, the RT-qPCR primers sequences used for detection of miR-498 and miR-944 are listed in Additional file 1: Table S2. The relative expression of target genes was calculated with the 2−∆∆CT method. All RT-qPCR primers were synthesized by Sangon Biotech (Shanghai, China).
Fluorescence in situ hybridization (FISH)
The FISH digoxigenin-labeled specific probe targeting circBRD7 was designed and synthesized by Sangon Biotech (Shanghai, China), and the sequence was 5’-TAGTTTGAAGTTATCCTGACTGTTCACAAG-3’. The FISH assay was performed according to the manufacturer’s instructions (GenePharma, China). First, cells were fixed in 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 at room temperature for 15 min. Thereafter, cells were hybridized with the FISH probe in hybridization buffer at 37 °C overnight. Finally, the cells were stained with DAPI, and photographed using a confocal microscope (Perkin-Elmer, Waltham, MA, USA).
Nuclear-cytoplasmic fractionation
The PARIS™ Kit Protein and RNA Isolation System (Invitrogen, CA, USA) were used to isolate the cytoplasmic and nuclear RNA of NPC cells according to the manufacturer’s instructions. Finally, reverse-transcribed RNA was used for RT-qPCR, and U6 and GAPDH were served as nuclear and cytoplasmic markers, respectively.
Actinomycin D treatment
NPC cells 5-8F and CNE2 were uniformly inoculated into 24-well plates. When the cell density reached 90%, actinomycetes D (Sigma, Missouri, USA) was added into the culture medium, and the solvent Dimethyl sulfoxide (DMSO; Sigma, MO, USA) was used as a negative control. After actinomycin D treatment for 0, 8, 16 and 24 h, cells were harvested and RNA was extracted. And then, reverse-transcribed of RNA was used for RT-qPCR.
CCK-8 and colony formation assays
For the CCK-8 assay, 1,000 cells/well were seeded into 96-well plates. CCK-8 reagent (Selleck, Houston, TX, USA) was added to each well and incubated for 2 h at the indicated time points (0, 1, 2, 3, 4 and 5 days). Then, the absorbance at 450 nm was measured with a microplate analyzer (Beckman, Brea, CA, USA). For the colony formation assay, 1,000 cells/well were plated into 6-well plates. The cells were allowed to grow for 1 to 2 weeks, and the colonies were observed with crystal violet staining.
Wound healing and transwell assays
Cells were seeded in 6-well plates, and then a 10 µL plastic pipette tip was used to create a straight scratch through the cell monolayer when the cells grew to approximately 90% confluence. Images of the scratch width were acquired at 0, 12 and 24 h after the initial scratch, and the area of cell migration was measured. For transwell assays, Matrigel (BD, Franklin Lakes, NJ, USA) was diluted using serum-free medium at a ratio of 8:1, and 20 μL of the diluted Matrigel was added to the transwell chamber (Millipore, NY, USA) and incubated for 3 h. Then 600 μL of medium containing 20% FBS was added to the lower chamber. After that, 5×104 cells were suspended in 200 μL of serum-free medium and added to the top chamber. After incubation at 37 °C for 48 h, the cells were fixed with 4% paraformaldehyde and stained with crystal violet, and the number of migrated cells was counted.
Luciferase reporter assay
The pGL3 enhancer and pRL-TK vectors were purchased from Promega (Fitchburg, WI, USA). To evaluate the effect of circBRD7 on BRD7 promoter activity, we constructed a recombinant reporter vector fused with the BRD7 promoter (pGL3 enhancer/BRD7 promoter). Cells were cotransfected with the pcDNA3.1(+) circRNA vector or circBRD7 overexpression plasmid, and the pRL-TK vector containing Renilla luciferase was used as an internal control. Cells were harvested following transfection for 48 h and analyzed by utilizing the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.
Chromatin immunoprecipitation assay (ChIP)
Cells were crosslinked with 1% formaldehyde at 37 °C for 10 min. And then, 250 μL 2.5 M glycine was added to terminate fixation. After that, cells were harvested and lysed and the supernatant was discarded after centrifugation. Nuclei lysis buffer was added to the precipitate and the chromatin was broken up into fragments of 100-500 bp by a BioRuptor sonicator (Diagenode). After centrifuged at 13000 rpm for 15 min, the supernatant obtained was whole cell extract (WCE). Subsequently, chromatin was immunoprecipitated with immunoglobulin IgG (Sigma) and anti-H3K27ac antibody (ABclonal, Wuhan, China) with the Protein A/G Magnetic Beads system (Selleck Chemicals, Houston, TX, USA) according to the manufacturer’s protocols. After overnight incubation with shaking at 4 °C, the supernatant was removed and the magnetic beads were washed with low salt buffer, high salt buffer, LiCl buffer and TE buffer successively. And then, Elution buffer was added to collect the product. The samples were incubated overnight at 65 °C to reverse cross-linking. Next, an equal volume of neutral phenol: chloroform: isopentyl alcohol (24:25:1) was added to the mixture to extract DNA. Finally, the collected DNA was used for subsequent RT-qPCR. The primers sequences used for ChIP-qPCR assay are listed in Table 3.
Western blot
The detailed method of western blot has been published [18,19]. Primary antibodies used in western blot are as follow: anti-BRD7 (Proteintech, Wuhan, China), anti-CDK4 (Proteintech, Wuhan, China), anti-GAPDH (Proteintech, Wuhan, China), anti-p21 (CST, MA, USA), anti-Snail (CST, MA, USA), anti-E-cadherin (CST, MA, USA), anti-N-cadherin (CST, MA, USA) and anti-Vimentin (Arigo, Taiwan, China). The protein bands were revealed by enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) following the manufacturer’s recommendations, and captured by chemiluminescence imaging systems (MiniChemi™ I, SAGECREATION, China).
In situ hybridization (ISH) and immunohistochemistry (IHC) staining
For ISH staining, the ISH kit was purchased from Boster Biological (Wuhan, China) and was used according to the manufacturer’s instructions. IHC staining was performed as previously described by the research group [19]. Sections were incubated with anti-BRD7 (Proteintech, Wuhan, China), anti-p21 (CST, MA, USA), anti-CDK4 (Proteintech, Wuhan, China), anti-Ki67 (Bioworld, Nanjing, China), anti-Vimentin (Arigo, Taiwan, China) and anti-E-cardherin (Abclonal, Wuhan, China). And MaxVisionTM HRP-Polymer anti-Mouse/Rabbit was incubated at room temperature for 30 min. After DAB staining, the nuclei were couterstained with hematoxylin (Solarbio, Beijing, China).
The results of ISH and IHC were analyzed according to the staining intensity and distribution of stained cells. The signal intensity and positive rate of positive cells in each sample were comprehensively scored, and the judgment criteria were as follows. The staining intensity was scored according to the following criteria, where 0 was defined as negative; 1 as weak; 2 as moderate; and 3 as strong. The distribution of stained cells was scored from 0 to 4, where no staining was scored as 0; positive rate of 1% to 25% was scored as 1; 26%-50% was scored as 2; 51% to 75% was recorded as 3 points, and 76-100% was recorded as 4 points. The sections were scored independently by two pathologists. The final score is the product of intensity score and positive rate score. The expression level was classified as low or high with the median total score as the cutoff [19,20].
In vivo nude mouse models
The five-week-old female BALB/C nude mice used were purchased from Hunan Slyke Jingda Experimental Animal Co., Ltd. All animals were fed in an SPF level barrier system of the Laboratory Animal Science Department of Central South University. For the xenograft tumor model, the nude mice were randomly divided into three groups (n = 5 per group): 5-8F/Ctrl, 5-8F/circBRD7, and 5-8F/circBRD7 plus siBRD7. A total of 5×106 transfected 5-8F cells in 150 μL of saline were inoculated into the left subcutaneous position of each nude mouse. Tumor size and volume were observed and measured every 3 days after injection. The tumor volume was calculated according to the following formula: volume = (length x width2) x 1/2. After 30 days of subcutaneous inoculation, all nude mice were euthanized. The tumors were removed and weighed, fixed with 4% paraformaldehyde, and preserved after paraffin embedding. For the lung metastatic tumor model, the nude mice were divided into 3 groups (n = 7 per group): 5-8F/Ctrl, 5-8F/circBRD7, and 5-8F/circBRD7 plus siBRD7. A total of 3×106 5-8F cells in 200 μL of saline were injected into nude mice via tail vein. Eight weeks later, the lung tissues were removed, and the number of nodules was observed and recorded. Then, the lung tissues were fixed with 4% paraformaldehyde, dehydrated by gradient alcohol, embedded in paraffin, and sectioned for H&E staining.
Statistical analysis
GraphPad Prism 7.0 software was applied for statistical analysis. All data in this study were expressed as mean ± standard error of the mean (SEM). Student's t-test was used to analyze the significant differences between two groups of data. One-way ANOVA was used to analyze differences among multiple sets of data. Correlations were evaluated by Pearson correlation analysis. P values less than 0.05 were considered statistically significant (ns, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Each experiment was conducted at least three times independently.