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RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation

  • Authors:
    • Hang Peng
    • Long Zhang
    • Fang Li
    • Xintao Jing
    • Jing Zhou
    • Li Cao
    • Cuixiang Xu
    • Jianhua Wang
    • Chen Huang
  • View Affiliations / Copyright

    Affiliations: Second Department of General Surgery, Shaanxi Provincial People's Hospital, Xi'an, Shaanxi 710068, P.R. China, Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China, Department of Cell Biology and Genetics/Key Laboratory of Environment and Genes Related to Diseases, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061, P.R. China, Shaanxi Provincial Key Laboratory of Infection and Immune Diseases, Shaanxi Provincial People's Hospital, Xi'an, Shaanxi 710068, P.R. China
    Copyright: © Peng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 131
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    Published online on: June 30, 2025
       https://doi.org/10.3892/ijmm.2025.5572
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Abstract

 RNA metabolism is an important post‑transcriptional regulatory mode in organisms, and its process is cooperatively regulated by a variety of RNA‑binding proteins. RNA binding motif protein 8A (RBM8A), a regulator of mRNA stability that is implicated in cancer progression, serves an important role in processes such as RNA splicing, transport, translation and decay. However, to the best of our knowledge, its role in the occurrence and development of gastric cancer (GC), as well as its biological functions and molecular mechanisms remain unclear. In the present study, RBM8A expression was on average 1.4‑fold higher (P<0.05), with a maximum log2 fold change of 1.4 (2.6‑fold increase), in GC tissues compared with adjacent normal tissues, as determined by multiplex immunohistochemical analysis of tissue microarrays. In vitro, transfection of RBM8A small interfering RNAs significantly suppressed the proliferation of AGS and HGC27 cells and enhanced apoptosis. Specifically, annexin V‑positive AGS cells exhibited a 2.9‑fold increase with siRBM8A‑1 transfection and a 1.9‑fold increase with siRBM8A‑2 transfection, while annexin V‑positive HGC27 cells exhibited a 2.3‑fold increase with siRBM8A‑1 transfection and a 1.8‑fold increase with siRBM8A‑2 transfection (P<0.05). Using MKN45 cell lines and subcutaneous xenograft models, the present study revealed that RBM8A knockdown reduced subcutaneous tumor growth in nude mice by 51.5% in terms of volume and 62.4% in terms of weight (P<0.05). In terms of the mechanism, integrated mRNA‑sequencing (seq) and RNA immunoprecipitation (RIP)‑seq identified BCL2 binding component 3 (BBC3), a well‑characterized pro‑apoptotic gene, as a direct target of RBM8A. Further results of RIP‑quantitative PCR, fluorescence in situ hybridization‑immunofluorescence and RNA pulldown indicated the direct interaction between RBM8A and BBC3 mRNA. Actinomycin D assays demonstrated that RBM8A promoted BBC3 mRNA degradation. Subsequently, the co‑immunoprecipitation assay showed that RBM8A interacted with UPF3B to jointly regulate the stability of BBC3 mRNA. In conclusion, RBM8A inhibited apoptosis and promoted GC progression by interacting with UPF3B, leading to degradation of the pro‑apoptotic gene BBC3 mRNA. These findings highlighted that interfering with RBM8A expression, or disrupting the interactions between RBM8A and BBC3 mRNA or between RBM8A and UPF3B could serve as potential therapeutic strategies for GC.
View Figures

Figure 1

RBM8A expression is upregulated in
GC. (A) mRNA expression levels of RBM8A in GC and normal tissues
from TCGA. An unpaired t-test was used for statistical analysis.
(B) RBM8A expression levels in GC and adjacent normal tissues in
TCGA. A paired t-test was used for statistical analysis. (C)
Disease-specific survival curves. The survival probability was
analyzed, and the log-rank test showed P=0.0361. (D) RBM8A and
PANCK were detected by multiplex immunohistochemistry in the GC
tissue microarray. The odd-numbered columns represent GC tissues,
and the even-numbered columns represent adjacent non-cancerous
tissues. Scale bar, 200 μm. (E) Statistical analysis of the
fluorescence intensity of RBM8A staining in the epithelial cells of
GC and adjacent non-cancerous tissues. A paired t-test was used for
statistical analysis. (F) Bar chart showing the log-transformed
fold change in RBM8A expression between GC tissues and adjacent
non-cancerous tissues. ***P<0.001. GC, gastric
cancer; PANCK, pan-cytokeratin; RBM8A, RNA binding motif protein
8A; TCGA, The Cancer Genome Atlas; TPM, transcripts per
million.

Figure 2

Knockdown of RBM8A inhibits gastric
cancer cell proliferation. (A) Reverse transcription-quantitative
PCR confirmed that siRBM8A was capable of effectively reducing the
RBM8A mRNA level. One-way ANOVA with Dunnett's multiple comparisons
test was used for statistical analysis. (B) Western blot analysis
of RBM8A protein levels in AGS and HGC27 cells transfected with
siRBM8A or siNC (negative control). (C) Densitometric
semi-quantification of RBM8A protein levels from the western blot
analysis in (B), normalized to GAPDH. One-way ANOVA with Dunnett's
multiple comparisons test was used for statistical analysis. (D)
CCK8 assay indicating that knockdown of RBM8A significantly
suppressed the proliferation of AGS cell at 72 h post-transfection.
Statistical analysis at the 72-h timepoint was performed using
one-way ANOVA with Dunnett's multiple comparisons test comparing
the siRBM8A-1 and siRBM8A-2 groups with the siNC group. (E) CCK8
assay showing that knockdown of RBM8A significantly suppressed
HGC27 cell proliferation at 72 h post-transfection. Statistical
analysis at the 72-h timepoint was performed using one-way ANOVA
with Dunnett's multiple comparisons test comparing the siRBM8A-1
and siRBM8A-2 groups with the siNC group. (F) Colony formation
assays revealed that knockdown of RBM8A significantly impaired the
colony formation of AGS and HGC27 cells. (G) Statistical analysis
of colony numbers in (F). One-way ANOVA with Dunnett's multiple
comparisons test was used for statistical analysis. (H) Flow
cytometry showed that knockdown of RBM8A significantly enhanced
apoptosis in AGS and HGC27 cells. One-way ANOVA with Dunnett's
multiple comparisons test was used for statistical analysis. (I)
Western blotting indicated that knockdown of RBM8A led to
upregulation of the protein levels of cleaved PARP1 and Bax.
Statistical analysis of the relative expression levels of cleaved
PARP1 and Bax is shown at the bottom. One-way ANOVA with Dunnett's
multiple comparisons test was used for statistical analysis. (J)
Knockdown of RBM8A had no marked effect on the migration of AGS and
HGC27 cells. One-way ANOVA with Dunnett's multiple comparisons test
was used for statistical analysis. Scale bar, 250 μm. Data
are presented as the mean ± SD. *P<0.05,
**P<0.01, ***P<0.001 vs. siNC. CCK8,
Cell Counting Kit-8; NC, negative control; ns, not significant; OD,
optical density; PARP1, poly(ADP-ribose) polymerase 1; RBM8A, RNA
binding motif protein 8A; sh, short hairpin RNA; si, small
interfering RNA.

Figure 3

RBM8A promotes gastric cancer cell
proliferation in vivo. (A) Nude mice 32 days after
inoculation with gastric cancer cells. Left, shRBM8A group; right,
shNC group. (B) Subcutaneous tumors in nude mice (n=3). (C) Volume
of the xenograft tumors following RBM8A knockdown. The volume of
tumors were estimated by measuring sizes every other day. A paired
t-test was used for statistical analysis. (D) Weight of harvested
xenograft tumors. A paired t-test was used for statistical
analysis. (E) Immunohistochemistry staining of xenograft tumors
showing that the protein levels of RBM8A were decreased in RBM8A
knockdown tumor tissues. Scale bar, 50 μm. Data are
presented as the mean ± SD. **P<0.01. NC, negative
control; RBM8A, RNA binding motif protein 8A; sh, short hairpin
RNA.

Figure 4

Analysis of mRNA expression profiles
after knockdown of RBM8A in AGS cells. (A) Heat map of DEGs. (B)
Volcano plot of mRNA expression profiles. (C) Gene Ontology
analysis of DEGs. A hypergeometric test with FDR correction was
used for statistical analysis. (D) Kyoto Encyclopedia of Genes and
Genomes analysis of DEGs. A hypergeometric test with FDR correction
was used for statistical analysis. DEG, differentially expressed
gene; FDR, false discovery rate; NC, negative control; p adj,
adjusted P-value; RBM8A, RNA binding motif protein 8A; si, small
interfering RNA.

Figure 5

RIP-seq identifying mRNAs that
directly bind with RBM8A. (A) RIP experiments with three biological
replicates (RBM8A 1, 2 and 3) identified mRNAs from 2,079 genes
that bind to RBM8A. (B) These genes are distributed across various
chromosomes. (C) Binding modes of RBM8A to mRNA and the proportion
of each mode. (D) Gene Ontology analysis of genes corresponding to
mRNAs that bind directly to RBM8A. A hypergeometric test with FDR
correction was used for statistical analysis. (E) Combined analysis
of mRNA-seq and RIP-seq to screen for target genes of RBM8A that
are related to cell apoptosis. (F) BBC3 mRNA expression levels were
significantly upregulated following knockdown of RBM8A. One-way
ANOVA with Dunnett's multiple comparisons test was used for
statistical analysis. Data are presented as the mean±SD.
*P<0.05, **P<0.01,
***P<0.001 vs. siNC. BBC3, BCL2 binding component 3;
chr, chromosome; FDR, false discovery rate; NC, negative control;
RBM8A, RNA binding motif protein 8A; RIP, RNA immunoprecipitation;
seq, sequencing; si, small interfering RNA; UTR, untranslated
region.

Figure 6

BBC3 mediates the inhibition of
apoptosis in GC cells by RBM8A. (A) siBBC3 knockdown efficiency
detected by reverse transcription-quantitative PCR. An unpaired
t-test was used for statistical analysis. (B) siBBC3 knockdown
efficiency detected by western blotting. (C) BBC3 mRNA expression
levels were measured after co-transfecting siRBM8A and siBBC3 into
AGS and HGC27 cells. One-way ANOVA with Tukey's Honestly
Significant Difference test was used for statistical analysis. (D)
CCK8 assay indicating that knockdown of BBC3 partially reversed the
inhibition of AGS cell proliferation caused by knockdown of RBM8A.
Statistical analysis at the 96-h timepoint included comparisons
between siRBM8A vs. siNC groups and siRBM8A+siBBC3 vs. siRBM8A
groups, performed using one-way ANOVA with Tukey's Honestly
Significant Difference test. (E) CCK8 assay indicating that
knockdown of BBC3 partially reversed the inhibition of HGC27 cell
proliferation caused by knockdown of RBM8A. Statistical analysis at
the 96-h timepoint included comparisons between siRBM8A vs. siNC
groups and siRBM8A+siBBC3 vs. siRBM8A groups, performed using
one-way ANOVA with Tukey's Honestly Significant Difference test.
(F) Flow cytometry showing that knockdown of BBC3 partially
reversed the promoting effect of RBM8A knockdown on the apoptosis
of GC cells. One-way ANOVA with Tukey's Honestly Significant
Difference test was used for statistical analysis. Data are
presented as the mean ± SD. *P<0.05,
***P<0.001. BBC3, BCL2 binding component 3; CCK8,
Cell Counting Kit-8; GC, gastric cancer; NC, negative control; OD,
optical density; RBM8A, RNA binding motif protein 8A; si, small
interfering RNA.

Figure 7

RBM8A interacts with BBC3 mRNA and
promotes its degradation. (A) Western blotting showed that RBM8A
antibody could effectively enrich RBM8A. (B) Quantitative PCR
showed that RBM8A antibody significantly enriched BBC3 mRNA in
gastric cancer cells. An unpaired t-test was used for statistical
analysis. (C) Agarose gel electrophoresis results showed that RBM8A
antibody enriched BBC3 mRNA. (D) RNA pulldown assay demonstrating
that the BBC3 mRNA probe enriched BBC3 mRNA. (E) RNA pulldown assay
showing that the BBC3 mRNA probe enriched RBM8A. (F) Fluorescence
in situ hybridization-immunofluorescence showed that there
was co-localization of RBM8A and BBC3 mRNA. Scale bar, 10
μm. (G) Act D assay showing that knockdown of RBM8A
inhibited BBC3 mRNA degradation in AGS cells. Statistical analysis
for all panels was performed using one-way ANOVA with Tukey's
Honestly Significant Difference test. For the right panel,
statistical analysis compared the fold change of BBC3 expression
post-Act D treatment relative to the pre-Act D treatment baseline
between the siNC and siRBM8A groups at multiple time points (2, 4
and 6 h). (H) Act D assay showing that knockdown of RBM8A inhibited
BBC3 mRNA degradation in HGC27 cells. Statistical analysis for all
panels was performed using one-way ANOVA with Tukey's Honestly
Significant Difference test. For the right panel, statistical
analysis compared the fold change of BBC3 expression post-Act D
treatment relative to the pre-Act D treatment baseline between the
siNC and siRBM8A groups at multiple time points (2, 4 and 6 h).
Data are presented as the mean ± SD. **P<0.01,
***P<0.001 vs. siNC or IgG. Act D, actinomycin D;
BBC3, BCL2 binding component 3; ctrl, control; IP,
immunoprecipitation; NC, negative control; RBM8A, RNA binding motif
protein 8A; si, small interfering RNA.

Figure 8

RBM8A regulates cell apoptosis by
interacting with UPF3B to modulate BBC3 mRNA levels. (A) Gene
Ontology analysis of RBM8A-interacting proteins detected by
CoIP-mass spectrometry. A hypergeometric test with FDR correction
was used for statistical analysis. (B) CoIP-western blotting
experiments showed that Flag antibody enriched RBM8A-3X Flag and
UPF3B in RBM8A-3X Flag-overexpressing AGS and HGC27 cells. (C)
CoIP-western blotting experiments showed that Flag antibody
enriched UPF3B-3X Flag and RBM8A in UPF3B-3X Flag-overexpressing
AGS and HGC27 cells. (D) qPCR examining the mRNA levels of RBM8A
following co-transfection with siRBM8A and UPF3B-OE. One-way ANOVA
with Tukey's Honestly Significant Difference test was used for
statistical analysis. (E) qPCR examining the mRNA levels of UPF3B
following co-transfection with siRBM8A and UPF3B-OE. One-way ANOVA
with Tukey's Honestly Significant Difference test was used for
statistical analysis. (F) qPCR results demonstrated that UPF3B
overexpression rescued the upregulation of BBC3 mRNA induced by
RBM8A knockdown in AGS and HGC27 cells. One-way ANOVA with Tukey's
Honestly Significant Difference test was used for statistical
analysis. (G) CCK8 assay indicating that UPF3B overexpression
partially reversed the inhibition of AGS cell proliferation caused
by knockdown of RBM8A. Statistical analysis at the 72-h timepoint
included comparisons between the siRBM8A + vector vs. siNC + vector
groups and siRBM8A+UPF3B-OE vs. siRBM8A + vector groups, performed
using one-way ANOVA with Tukey's Honestly Significant Difference
test. (H) CCK8 assay indicating that UPF3B overexpression partially
reversed the inhibition of HGC27 cell proliferation caused by
knockdown of RBM8A. Statistical analysis at the 72-h timepoint
included comparisons between the siRBM8A + vector vs. siNC + vector
groups and siRBM8A + UPF3B-OE vs. siRBM8A + vector groups,
performed using one-way ANOVA with Tukey's Honestly Significant
Difference test. (I) Flow cytometry results showed that UPF3B
overexpression partially reversed the promoting effect of RBM8A
knockdown on the apoptosis of gastric cancer cells. One-way ANOVA
with Tukey's Honestly Significant Difference test was used for
statistical analysis. Data are presented as the mean ± SD.
*P<0.05, **P<0.01,
***P<0.001. BBC3, BCL2 binding component 3; CCK8,
Cell Counting Kit-8; CoIP, co-immunoprecipitation; FDR, false
discovery rate; NC, negative control; ns, not significant; OD,
optical density; OE, overexpression plasmid; qPCR, quantitative
PCR; RBM8A, RNA binding motif protein 8A; si, small interfering
RNA.
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Copy and paste a formatted citation
Spandidos Publications style
Peng H, Zhang L, Li F, Jing X, Zhou J, Cao L, Xu C, Wang J and Huang C: RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation. Int J Mol Med 56: 131, 2025.
APA
Peng, H., Zhang, L., Li, F., Jing, X., Zhou, J., Cao, L. ... Huang, C. (2025). RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation. International Journal of Molecular Medicine, 56, 131. https://doi.org/10.3892/ijmm.2025.5572
MLA
Peng, H., Zhang, L., Li, F., Jing, X., Zhou, J., Cao, L., Xu, C., Wang, J., Huang, C."RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation". International Journal of Molecular Medicine 56.3 (2025): 131.
Chicago
Peng, H., Zhang, L., Li, F., Jing, X., Zhou, J., Cao, L., Xu, C., Wang, J., Huang, C."RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation". International Journal of Molecular Medicine 56, no. 3 (2025): 131. https://doi.org/10.3892/ijmm.2025.5572
Copy and paste a formatted citation
x
Spandidos Publications style
Peng H, Zhang L, Li F, Jing X, Zhou J, Cao L, Xu C, Wang J and Huang C: RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation. Int J Mol Med 56: 131, 2025.
APA
Peng, H., Zhang, L., Li, F., Jing, X., Zhou, J., Cao, L. ... Huang, C. (2025). RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation. International Journal of Molecular Medicine, 56, 131. https://doi.org/10.3892/ijmm.2025.5572
MLA
Peng, H., Zhang, L., Li, F., Jing, X., Zhou, J., Cao, L., Xu, C., Wang, J., Huang, C."RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation". International Journal of Molecular Medicine 56.3 (2025): 131.
Chicago
Peng, H., Zhang, L., Li, F., Jing, X., Zhou, J., Cao, L., Xu, C., Wang, J., Huang, C."RBM8A promotes gastric cancer progression by binding with UPF3B to induce BBC3 mRNA degradation". International Journal of Molecular Medicine 56, no. 3 (2025): 131. https://doi.org/10.3892/ijmm.2025.5572
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