RBFOX2/GOLIM4 Splicing Axis Activates Vesicular Transport Pathway to Promote Nasopharyngeal Carcinogenesis

Abstract

20-30% of patients with nasopharyngeal carcinoma (NPC) develop distant metastasis or recurrence leading to poor survival, of which the underlying key molecular events have yet to be addressed. Here alternative splicing events in 85 NPC samples are profiled using transcriptome analysis and it is revealed that the long isoform of GOLIM4 (-L) with exon-7 is highly expressed in NPC and associated with poor prognosis. Lines of evidence demonstrate the pro-tumorigenic function of GOLIM4-L in NPC cells. It is further revealed that RBFOX2 binds to a GGAA motif in exon-7 and promotes its inclusion forming GOLIM4-L. RBFOX2 knockdown suppresses the tumorigenesis of NPC cells, phenocopying GOLIM4-L knockdown, which is significantly rescued by GOLIM4-L overexpression. High expression of RBFOX2 is correlated with the exon-7 inclusion of GOLIM4 in NPC biopsies and associated with worse prognosis. It is observed that RBFOX2 and GOLIM4 can influence vesicle-mediated transport through maintaining the organization of Golgi apparatus. Finally, it is revealed that RAB26 interacts with GOLIM4 and mediates its tumorigenic potentials in NPC cells. Taken together, the findings provide insights into how alternative splicing contributes to NPC development, by highlighting a functional link between GOLIM4-L and its splicing regulator RBFOX2 activating vesicle-mediated transport involving RAB26.

Keywords: GOLIM4; RAB26; RBFOX2; alternative splicing; nasopharyngeal carcinoma.

Figure 1

Identification of alternative splicing of GOLIM4 related to NPC progression. A) Gene ontology analysis of aberrant alternative splicing events in NPC tissues (n = 85) and control samples (n = 10). B) Heatmap of percent spliced‐in index (PSI) for 63 splicing events in the above samples. C) High PSI of GOLIM4 with the exon‐7 in the above NPC samples. D) Schematic diagram of GOLIM4 splicing variants. Constitutive exons are shown as grey and black boxes, with black, grey, and red boxes for untranslated regions, coding regions, and the alternative exon‐7, respectively. E) RT‐PCR showed the transcription of the splicing variants of GOLIM4 in NPC biopsies (T, n = 20) and control rhinitis tissues (N, n = 19). PSI value of each sample was shown at the bottom. ACTIN was used as control. F) qRT‐PCR showed the transcription levels of GOLIM4 variants in samples described in (E). G,H) Kaplan–Meier survival curves of disease‐free survival for patients with NPC grouped by PSI level of GOLIM4‐L and the total mRNA expression level of GOLIM4. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

GOLIM4‐L promotes proliferation and tumorigenesis of NPC cells in vitro and in vivo. A) Human NPC cell lines S26 and 5–8F were transiently transfected with two siRNAs against GOLIM4‐L variant. GOLIM4‐L knockdown was assessed using qRT‐PCR. B) Colony formation assays were performed with cells described in (A). Three independent experiments were performed, and representative cells stained with crystal violet were shown (left). The number of focal adhesions was qualified, and results were shown as mean ± SD (Standard Deviation; right). C) Representative images of EdU staining assay for cells described in (A) (left). Data at the right are presented as mean ± SD. D) Cell growth curves were determined for S26 and 5–8F cells infected with lentivirus‐expressing GOLIM4‐L/S or empty vector control as indicated. E) Tumor growth of xenograft derived from S26 cells infected with sh‐GOLIM4‐L#1, sh‐GOLIM4‐L#2, and sh‐Luci lentivirus at different time courses. Tumor volumes were measured every week. F) Tumor size (upper) and weight (bottom) for the xenograft excised from (E). G) Fluorescent staining of Ki67 and DAPI in formalin‐fixed paraffin‐embedded tumor samples derived from (F). H) Tumor growth of xenograft derived from S26 cells with stable overexpression of GOLIM4‐L or GOLIM4‐S, or with control vector at different time courses. I) Tumor size (left) and weight (right) for the xenograft excised from (F). Scale bar, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

RBFOX2 promotes the inclusion of GOLIM4 exon‐7 through binding to the alternative exon. A) Correlation between the PSI of GOLIM4‐L and the mRNA level of RBFOX2 in NPC tissues (n = 85) and control samples (n = 10). Correlation coefficient R squared and p values were based on Pearson's correlation test. B) RT‐PCR examination of GOLIM4 variants in S26 cells with RBFOX2 knockdown using siRNAs or overexpression with lentivirus. C) Schematic diagram of RBFOX2 domains and constructs of two RBFOX2 mutants, ΔRRM (deleting RRM) and ΔAla‐rich (deleting Ala‐rich). Both the mutants and RBFOX2‐WT were HA tagged. D) Western blotting assays showed the expression of RBFOX2, HA‐tagged proteins, and ACTIN in 293T cells. Cells were transiently transfected with plasmids described in (C) and empty vector as control. E) Verification of direct binding between proteins as indicated on top and endogenous GOLIM4 RNA fragments using CLIP assays. F) Schematic diagram of GOLIM4 minigene constructs. RT‐PCR was performed to examine the splicing pattern of GOLIM4 in 293T cells, which were co‐transfected with GOLIM4 minigene plasmids along with DNA constructs for siRNAs targeting RBFOX2 or control, as well as wildtype or mutant RBFOX2 as indicated on top. G) Schematic diagram of GOLIM4 minigene with indicated deletions tiling exon‐7 were generated (upper). RT‐PCR examined the transcription of GOLIM4 variants in 293 T cells (bottom), which were co‐transfected with si‐RBFOX2 (KD, as Knockdown) or si‐NC (Con, as Control) along with GOLIM4 mutant plasmids. H) RT‐PCR showed in vivo splicing of GOLIM4 mutant constructs in 293T cells, which were co‐transfected with GOLIM4 mutant plasmids indicated to the left along with DNA constructs for siRNAs targeting RBFOX2 or control, as well as wildtype or mutant RBFOX2 as indicated on top.

Figure 4

Overexpression of GOLIM4‐L rescues the inhibitory effects of RBFOX2 knockdown on NPC cells. A) Western blotting assay showed the expression of RBFOX2 and ACTIN in S26 and 5–8F cells transfected with sh‐RBFOX2‐1, sh‐RBFOX2‐2, or sh‐Luci lentivirus. B) Cell proliferation assays were performed using cells described in (A). C) Representative images of EdU staining assays (left) and the percentage of EdU positive cells (right) for cell lines described in (A). Data at the right are presented as mean ± SD. D) Colony formation assay was performed with S26 cells expressing shRBFOX2 or sh‐Luci (control) that infected with lentivirus expressing GOLIM4‐L, GOLIM4‐S or empty vectors. Quantification of colony numbers was shown at the bottom. Three independent experiments were performed, and error bars indicate SD of mean. E) Representative images of EdU staining assays (left) for cells described in (D) and corresponding statistics are shown at the right. F) Tumor growth of xenograft derived from S26 cells described in (D) at different time courses. G) Tumor size (left) and tumor weight (right) for xenograft excised from (F). H) Fluorescent staining of Ki67 and DAPI in formalin‐fixed paraffin‐embedded tumor samples derived from (G). Scale bar, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Expression of RBFOX2 in NPC and its association with prognosis of patients with NPC. A) Transcriptome analysis showed the mRNA expression of RBFOX2 in NPC tissues (n = 85) and control samples (n = 10). B) qRT‐PCR showed the transcription level of RBFOX2 (in relative to ACTIN) in another independent sample collection of NPC biopsies (n = 20) and control tissues (n = 19). C) Correlation between the PSI of GOLIM4‐L and the mRNA level RBOFX2 in samples described in (B). D) Western blotting assays showed the protein expression of RBFOX2 in samples described in (B). ACTIN was used as control. E) Western blotting assays showed the protein levels of RBFOX2 and ACTIN in NPC cell lines and normal nasopharyngeal epithelium cells (NP69). F) Representative images of immunohistochemistry (IHC) assay for formalin‐fixed paraffin‐embedded NPC tissues (n = 98) with RBFOX2 antibody. Scale bar, 100 µm. G,H) Kaplan–Meier survival curves of overall and disease‐free survival in patients with NPC as described in (F) stratified by the protein level of RBFOX2. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Pathways regulated by RBFOX2 and GOLIM4‐L in NPC cells. A) RBFOX2‐ affected alternative splicing events in S26 cells stably expressing RBFOX2‐shRNA and control shRNA (sh‐Luci). The events are classified into five categories: Cassette, mutually exclusive exon (MXE), alternative 5′‐splice site (A5SS), alternative 3′‐ splice site (A3SS) and retained intron (RI). B) Heatmap of alternative splicing events affected by RBFOX2 in above cells. The data were sorted by the mean value of WT (sh‐Luci) and KD (sh‐RBFXO2) groups. Red and blue indicated exon inclusion or exclusion, respectively. C) RNA sequencing reads mapping to GOLIM4 in above cells. D) RT‐PCR examination of GOLIM4 variants in S26 and 5–8F cells with RBFOX2 knockdown using shRNA. E) Significant gene ontology pathways affected by RBFOX2‐targeted splicing events. F) Significant biological processes involving genes with differential expression in S26 cells with GOLIM4‐L knockdown. G) Immunofluorescent staining showed the co‐localization of endogenous GOLIM4 (green) and GM130 (red) in S26 and 293T cells. Scale bar, 100 µm. H) Immunofluorescent staining of endogenous GM130 (red) together with RBFOX2 (green) or GOLIM4 (green) in S26 cells transfected with RBFOX2, GOLIM4‐L siRNAs or control siRNAs indicated on top. Scale bar, 100 µm. I) Electron microscopy showed the ultrastructure of Golgi in S26 cells transfected with siRNAs of RBFOX2, GOLIM4‐L, or control. Scale bar, 200 nm.

Figure 7

GOLIM4‐L binds and recruits RAB26 to mediate vesicle transportation. A) Immunofluorescent staining showed the localization of endogenous GM130 (green) and RAB26 (red), as well as colocalizations of exogenous RAB26 with Flag‐tag (red) and endogenous GOLIM4 (green) or exogenous GOLIM4‐L with HA‐tag (green). Scale bar, 100 µm. DNA constructs indicated on top were transiently introduced in 293T cells and whole cell lysis (input) or proteins immunoprecipitated (IP) with B) anti‐HA or C) anti‐FLAG antibody were immunoblotted with antibodies indicated at the left. Immunofluorescent staining of endogenous GM130 (red) and RAB26 (green) in S26 cells transfected with D) GOLIM4‐L siRNAs, or E) RAB26 siRNAs, or control siRNA. Scale bar, 100 µm. F) Electron microscopy showed the ultrastructure of Golgi in S26 cells described in (E). Scale bar, 200 nm. G) Cell proliferation assays were performed using S26 cells transfected with RAB26 siRNAs or control siRNA. H) Colony formation assays were performed with cells described in (E). Quantification of colony numbers was shown at the right. I) Representative images of EdU staining assays (left) for cells described in (E) and corresponding statistics are shown at the right. Scale bar, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

Schematic diagram showing a proposed model for the interactions among RBFOX2, GOLIM4, and RAB26 in NPC progression. RBFOX2 mediates the alternative splicing of the exon‐7 of GOLIM4 through binding to GGAA sequence in the exon, which generates the long isoform of GOLIM4‐L. GOLIM4‐L promotes tumorigenesis of NPC, likely through vesicle‐mediated transport pathway involving recruitment of RAB26.