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. 2024 Jan;115(1):139-154.
doi: 10.1111/cas.15998. Epub 2023 Nov 8.

CircBRD7 attenuates tumor growth and metastasis in nasopharyngeal carcinoma via epigenetic activation of its host gene

Jianxia Wei  1   2   3 , Mengna Li  1   2   3 , Shipeng Chen  1   2   3 , Changning Xue  1   2   3 , Lemei Zheng  1   2   3 , Yumei Duan  1   2   3 , Hongyu Deng  1 , Songqing Fan  4 , Wei Xiong  1   2   3 , Ming Zhou  1   2   3
Affiliations

CircBRD7 attenuates tumor growth and metastasis in nasopharyngeal carcinoma via epigenetic activation of its host gene

Jianxia Wei et al. Cancer Sci. 2024 Jan.

Abstract

BRD7 was identified as a tumor suppressor in nasopharyngeal carcinoma (NPC). Circular RNAs (CircRNAs) are involved in the occurrence and development of NPC as oncogenes or tumor suppressors. However, the function and mechanism of the circular RNA forms derived from BRD7 in NPC are not well understood. In this study, we first identified that circBRD7 was a novel circRNA derived from BRD7 that inhibited cell proliferation, migration, invasion of NPC cells, as well as the xenograft tumor growth and metastasis in vivo. Mechanistically, circBRD7 promoted the transcriptional activation and expression of BRD7 by enhancing the enrichment of histone 3 lysine 27 acetylation (H3K27ac) in the promoter region of its host gene BRD7, and BRD7 promoted the formation of circBRD7. Therefore, circBRD7 formed a positive feedback loop with BRD7 to inhibit NPC development and progression. Moreover, restoration of BRD7 expression rescued the inhibitory effect of circBRD7 on the malignant progression of NPC. In addition, circBRD7 demonstrated low expression in NPC tissues, which was positively correlated with BRD7 expression and negatively correlated with the clinical stage of NPC patients. Taken together, circBRD7 attenuates the tumor growth and metastasis of NPC by forming a positive feedback loop with its host gene BRD7, and targeting the circBRD7/BRD7 axis is a promising strategy for the clinical diagnosis and treatment of NPC.

Keywords: BRD7; circBRD7; host gene; nasopharyngeal carcinoma; tumor progression.

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Conflict of interest statement

The authors have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
CircBRD7 was identified as a circular RNA encoded by its host gene BRD7. (A) Agarose gel electrophoresis identified circ_0039345 fragments amplified in complementary DNA (cDNA) and genomic DNA (gDNA) using divergent primer and convergent primer. (B) The back‐splicing junction and total length of circBRD7 were validated by Sanger sequencing. (C) RT‐qPCR was used to detect the expression of circBRD7 in nine nasopharyngeal carcinoma (NPC) cell lines and normal NPE cell NP69. Actin served as an internal control. (D) Intracellular localization of circBRD7 in 5‐8F and CNE2 cells was detected by FISH. Nuclei were stained with DAPI. Scale bar, 20 μm. (E) The expression of circBRD7 in cytoplasm and nucleus was detected by nucleo‐cytoplasmic separation assay. GAPDH, cytoplasmic marker; U6, nuclear marker. (F) The stability of circBRD7 and BRD7 was detected in actinomycin D‐treated 5‐8F and CNE2 cells using RT‐qPCR. Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001.
FIGURE 2
FIGURE 2
CircBRD7 inhibits cell proliferation and cell cycle progression of nasopharyngeal carcinoma (NPC) cells. (A) RT‐qPCR assay was used to detect the expression efficiency of circBRD7 overexpressed vector in 5‐8F and CNE2 cells. (B, C) Cell counting kit‐8 (CCK‐8) (B) and colony formation (C) assays were used to detect the effect of circBRD7 overexpression on the proliferation and colony formation abilities of 5‐8F and CNE2 cells. (D, E) Flow cytometry was performed to detect the effect of circBRD7 overexpression on the cell cycle process (D) and cell apoptosis (E) of NPC cells. (F) Western blot assay was used to detect the expression of cyclin‐related molecules CDK4, p21, and apoptosis‐related molecules t‐PARP and c‐PARP, with GAPDH as normalized control. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.
FIGURE 3
FIGURE 3
CircBRD7 inhibits cell migration, invasion, and epithelial–mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC) cells. (A, B) Representative and quantified results of the wound healing (A) and transwell (B) assays in 5‐8F and CNE2 cells transfected with circBRD7 overexpression plasmid or blank vector. (C) Western blot was used to analyze the expression of EMT‐related molecules E‐cadherin, N‐cadherin, Vimentin, and Snail before and after circBRD7 overexpression. Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001; ns, no significance.
FIGURE 4
FIGURE 4
CircBRD7 promotes the transcriptional activation of BRD7. (A) BRD7 expression was detected by RT‐qPCR and western blot assays under circBRD7 overexpression. (B) RT‐qPCR and western blot experiments were performed to detect the effect of silencing circBRD7 on BRD7 expression. (C, D) A dual luciferase reporter assay was performed to detect the luciferase activity of BRD7 promoter (C) and the effect of circBRD7 on BRD7 promoter activity (D). (E) ChIP‐qPCR was used to determine the enrichment of H3K27ac in different regions of the BRD7 promoter and the effect of circBRD7 on the modification level of H3K27ac in BRD7 promoter fragments (−2000 to −1220 bp). DNA isolated from immunoprecipitate was amplified by qPCR, and normal rabbit IgG was used as negative control. H3K27ac, histone 3 lysine 27 acetylation. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.
FIGURE 5
FIGURE 5
Restoring BRD7 expression partially reverses the inhibitory effect of circBRD7 on the malignant phenotype of nasopharyngeal carcinoma (NPC) cells. (A) RT‐qPCR and western blot were used to detect the expression of circBRD7 and BRD7 in each group. (B) The cell counting kit‐8 (CCK‐8) assay was used to detect the effects of circBRD7 overexpression and restoring BRD7 expression on the proliferation of NPC cells 5‐8F and CNE2. (C–E) Representative and quantified results of the colony formation (C), wound healing (D), and transwell (E) assays in 5‐8F and CNE2 cells. (F) Western blot was performed to detect the alterations of expression of cell cycle‐, apoptosis‐ and epithelial–mesenchymal transition (EMT)‐related molecules before and after BRD7 restoration. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.
FIGURE 6
FIGURE 6
CircBRD7 attenuates tumor growth and metastasis in vivo by promoting its host gene expression. (A) Subcutaneous tumor growth curves in nude mice. (B) The images of nude mice in each group were photographed. (C) Images of subcutaneous tumor masses in each group after the nude mice were killed. (D) The tumor weights of each group (n = 5 per group) were measured. (E) Representative images of immunohistochemical staining (IHC) staining for the expression of BRD7 and some cell proliferation, cell cycle, and epithelial–mesenchymal transition (EMT)‐related molecules in xenograft tumor tissues (400×, scale bar, 50 μm). (F) Representative image of visible nodules on the lung surface of the metastatic tumor model in nude mice, with the red arrow representing pulmonary nodules. (G) The number of metastatic nodules on each lung surface was quantified, and each data point represents a nude mouse (n = 7 per group). (H) Typical images of lung metastatic foci after H&E staining of lung tissue sections. Red arrows represent pulmonary nodules. (I) Quantitative statistical histogram of the number of pulmonary nodules in lung tissue sections of four nude mice. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
FIGURE 7
FIGURE 7
Expression and clinical correlation of circBRD7 and BRD7 in biopsy tissues of nasopharyngeal carcinoma (NPC) at different stages. (A, B) Representative images (A) and statistical quantification charts (B) of in situ hybridization staining (ISH) and immunohistochemical staining (IHC) experiments to detect the expression of circBRD7 and BRD7 in noncancerous nasopharyngeal tissues and biopsies of NPC with different clinical TNM stages. (C) Correlation analysis between the expression of circBRD7 and its host gene BRD7 expression. (D) RT‐qPCR was used to detect the expression of circBRD7 in eight noncancerous nasopharyngeal tissues and 30 biopsy tissues of NPC. (E) RT‐qPCR was used to detect the expression of BRD7 in the same eight noncancerous nasopharyngeal tissues and 30 biopsy tissues of NPC. (F) Correlation analysis between the expression of circBRD7 and its host gene BRD7 expression in another set of independent fresh biopsies. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
FIGURE 8
FIGURE 8
Schematic diagram of the molecular mechanism of circBRD7 inhibiting the malignant progression of nasopharyngeal carcinoma (NPC). CircBRD7 is a circRNA formed by reverse splicing of exons 6–8 of the BRD7 and plays a tumor suppressive role in NPC by inhibiting the proliferation, migration, and invasion of NPC cells as well as xenograft tumor growth and metastasis. Mechanistically, circBRD7 enhanced the transcriptional activity of BRD7 by enhancing the enrichment of H3K27ac in its promoter region, and BRD7 promoted the expression of circBRD7. CircBRD7/BRD7 formed a positive feedback loop, thus inhibiting carcinogenesis and tumor progression in NPC. H3, histone 3; Ac, acetylation; EMT, epithelial‐mesenchymal transition.
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