Fig 2: BRD7 negatively regulates the expression of BIRC2 by inhibiting the activation of the BIRC2 enhancer.A ChIP-PCR assays using antibody specific for Flag was performed to validate the BRD7-binding sites in the 6th intron of BIRC2, while a 400-bp fragment of the BIRC2 potential promoter region without BRD7 binding site was used as a negative control. B Western blotting using antibodies against BRD7 and BIRC2 was performed to confirm BRD7 and BIRC2 protein levels. GAPDH served as an internal control. C qRT-PCR assays were used to confirm BRD7 and BIRC2 mRNA levels. GAPDH served as an internal control. D The dual-luciferase reporter assays was performed to determine the effect of BRD7 on the promoter activity of BIRC2. E ChIP-PCR assay using antibodies specific for H3K4me1 was used to validate the H3K4me1 enrichment at the B7BS, and B7BS was divided into four PCR fragments of about 200 bp, each fragments was amplified with each pair of primers. (F) and (G) The dual-luciferase reporter assays were used to determine the enhancer activities of B7BS and mut-B7BS and the effect of BRD7 on their activities. The error bars are presented as the mean ± SD. ***P < 0.001; NS, no significance. All experiments were performed in triplicate.

Fig 3: BIRC2 promotes tumor progression and functions as a candidate oncogene in nasopharyngeal carcinoma.A Western blotting was performed to detect the BIRC2 protein level using antibody against BIRC2, and GAPDH served as an internal control. B CCK-8 assay was performed to detect the cell viability of 5-8 F and HNE1 cells with stable overexpression of BIRC2. C Colony-forming assay was used to detect the cell growth of 5-8 F and HNE1 cells with stable overexpression of BIRC2. D Flow cytometry analysis of cell cycle distribution, and the representative (left) and statistical results (right) were shown. E Flow cytometry analysis of cell apoptosis via Annexin V-FITC and PI double staining, and the representative (left) and statistical results (right) were shown. F Scratch wound healing analysis of cell migration in 5-8 F and HNE1 cells stably transfected with BIRC2 expression plasmid or control. G Matrigel invasion analysis of cell invasive capabilities in 5-8 F and HNE1 cells stably transfected with BIRC2 expression plasmid or control. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significance. All experiments were performed in triplicate.

Fig 4: Depletion of BIRC2 by siRNA promotes cell proliferation and metastasis in NPC cell lines.A 5-8 F or HNE1 cells were transfected with scrambled siRNA (negative control) and BIRC2 siRNA #1, #2, respectively. Western blotting was used to analyze the silencing efficiency of BIRC2, and GAPDH served as an internal control. (B) and (C) Cell growth was measured by the CCK-8 assays and colony-forming assays of 5-8 F and HNE1 with transfection of BIRC2 siRNAs or negative control. D Cell migration assay was performed with the scratch wound healing analysis. E Matrigel invasion analysis of cell invasive capabilities in 5-8 F and HNE1 with transfection of BIRC2 siRNAs or negative control. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significance. All experiments were performed in triplicate.

Fig 5: BRD7 expression was negatively correlated with BIRC2 in clinical NP and NPC tissues.A Representative image of BRD7 and BIRC2 expression in noncancerous nasopharyngeal tissues and different clinical TNM stages of NPC detected by IHC. B Statistical diagram of BRD7 and BIRC2 expression in noncancerous nasopharyngeal tissues and different clinical TNM stages of NPC. C Correlation between BRD7 expression and BIRC2 expression. D A schematic map showing the tumor-suppressive mechanism of BRD7 that occurs by repressing the enhancer activity and expression of BIRC2 in NPC. Red represents upregulated genes, and blue represents downregulated genes. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.