BCL6-SPECC1L: A Novel Fusion Gene in Nasopharyngeal Carcinoma

Abstract

Background: Nasopharyngeal carcinomas (NPCs) are malignant tumors originating from the lining epithelium of the nasopharynx. Fusion genes have been confirmed to play important roles in the occurrence and development of various malignant tumors, but the role of fusion genes in NPC is poorly understood. We aimed to explore new fusion genes that promote the occurrence and development of NPC. Methods: RNA-seq was used to search for interchromosomal translocations in 18 NPC tissues. Polymerase chain reaction (PCR) and Sanger sequencing were applied to verify the presence of BCL6-SPECC1L (BS); quantitative PCR (qPCR) and Western blotting were used to measure the expression level of BCL-6 in NPC cells; MTT and in vivo tumorigenesis assays were applied to evaluate the cell proliferation ability; immunofluorescence assays were used to determine the cellular localization of BCL6 and BS; and a luciferase reporter assay was performed to evaluate the ability of BCL6 and BS to inhibit transcription. Results: BS was present in 5.34% (11/206) of primary NPC biopsies and 2.13% (1/47) of head and neck cancer biopsies. The expression of BCL6 was downregulated in NPC, and silencing of endogenous BCL6 promoted NPC cell proliferation in vitro. Overexpression of BCL6 but not BS inhibited the growth of NPC cells in vivo and in vitro. Mechanistically, BCL6 localized in the nucleus can inhibit the G1/S transition to suppress the growth of NPC cells. However, after the fusion of BCL6 and SPECC1L, the product cannot localize to the nucleus, and the transcriptional inhibitory function of BCL6 is abolished, eventually abolishing its tumor suppressor effect and leading to the development of NPC. Conclusion: BS is a novel fusion gene in NPC that may play an important role in the occurrence and development of this cancer. The clinical significance of the BS fusion gene needs further elucidation.

Keywords: BCL6; RNA-seq; SPECC1L; fusion gene; nasopharyngeal carcinoma.

Figure 1.

Discovery of the BS fusion gene in NPC. (A) Detection of the BS fusion gene using transcriptome sequencing. The reads are aligned across the junctions of the predicted fusion transcripts. (B) The existence of BS at the transcriptional level (left) and genomic level (right) in NPC tissue 271 was confirmed by RT-PCR. (C) Identification of the fusion breakpoint at the transcriptional level using Sanger sequencing. (D) Identification of the fusion breakpoint at the genomic level using Sanger sequencing. The black bar indicates the 4 bp (TTCC) microhomologous region in the junction. (E) RNA structure (left) and protein structure (right) of the predicted fusion. All experiments were repeated 3 times and representative results are shown.

Figure 2.

BCL6 is a tumor suppressor gene in NPC. The expression level of BCL6 in various NPC cell lines was measured by qPCR (2A) and WB (2B). (C) In 2 GEO datasets, BCL6 was downregulated in NPC. MTT assays of HNE1 cells (D) and HONE1 cells (E) seeded 24 h after transfection with siRNA and then cultured for the indicated period; the siRNA interference efficiency is shown on the left. n = 6 wells per group. * refers to differences between NC and siBCL6-1 or siBCL6-2 (* P < .05; ** P < .01; and *** P < .001 (two-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test)). Growth curve (F) and images (G) of tumors from nude mice inoculated with HONE1 cells stably expressing BCL6 or pMSCV (vector). All in vitro experiments were repeated 3 times and representative results are shown.

Figure 3.

Effect of the fusion gene on the anticancer effect of BCL6 and the underlying mechanism. MTT assays of HNE1 cells (A) and HONE1 cells (B) stably expressing BCL6 or BS. MTT assays of HNE1 cells (C) and HONE1 cells (D) seeded 24 h after transfection with SPECC1L siRNA and then cultured for the indicated period. n = 6 wells per group. * refers to differences between vector and BCL6 or BS or between NC and siSPECC1L-1 or siSPECC1L-2 (* P < .05; ** P < .01; and *** P < .001 (two-way ANOVA followed by the Bonferroni post hoc test). (C) Immunofluorescence staining was performed to detect the localization of BCL6 and BS in NPC cells. Nuclei were counterstained with DAPI. (D) The transcriptional inhibition ability of BCL6, BS, and BCL6^1-513 was identified by a luciferase reporter assay. * refers to differences between BCL6 and BS or BCL6^1-513 (* P < .05; ** P < .01; and *** P < .001 (ANOVA followed by the Bonferroni post-hoc test). All experiments were repeated 3 times and representative results are shown.

Figure 4.

Representative Western blot images showing p21, p27, and GAPDH expression in HNE1 and HONE1 cells with BCL6 or BS overexpression, or with knockdown of BCL6 expression. The experiment was repeated 3 times and representative results are shown (A). The blots were quantified using Image Lab software (Bio-Rad, version 6.1), and paired T-test was used for statistical test (B and C).