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Article Open Access

DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells

  • Authors:
    • Jiaxing Sun
    • Xiaoxing Huang
    • Xingruo Zeng
    • Yufei Lei
    • Hengjing He
    • Zimeng Wei
    • Di Xiao
    • Qiuping Zhang
    • Xinran Li
    • Fuling Zhou
    • Liang Shao
  • View Affiliations / Copyright

    Affiliations: Department of Hematology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Blood Transfusion, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Immunology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China
    Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 149
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    Published online on: September 3, 2025
       https://doi.org/10.3892/or.2025.8982
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Abstract

T-cell acute lymphoblastic leukemia (T‑ALL) is an aggressive hematological malignancy. The poor prognosis of T‑ALL is closely associated with extensive leukemic infiltration into critical organs. Therefore, the mechanism underlying T‑ALL infiltration is worth investigating. Databases and clinical samples were utilized to examine drebrin 1 (DBN1) expression in T‑ALL. DBN1 knockdown cell lines were established by lentivirus transfection, and cell migration and invasion were examined using Transwell and Matrigel‑Transwell assays. The molecular mechanism was investigated by RNA sequencing and further validated at the molecular level. Reverse transcription‑quantitative PCR and western blotting were employed to examine the expression of downstream molecules following DBN1 knockdown, with subsequent rescue experiments. DBN1‑targeting microRNA (miR) predicted using bioinformatics websites was confirmed using dual‑luciferase assays. In T‑ALL cells, miRNA mimics transfection enabled functional validation, and investigations into the underlying molecular mechanisms encompassing rescue experiments. Clinical samples and publicly available databases revealed that DBN1 was upregulated in patients with T‑ALL patients. DBN1 knockdown significantly decreased the migration and invasion of T‑ALL cells in vitro. RNA sequencing revealed that downregulation of DBN1 could reduce Grb2‑associated binding protein 2 (GAB2) expression. Western blotting revealed that GAB2 expression, and PI3K/AKT and MAPK/ERK signaling were decreased in DBN1‑knockdown cells. GAB2 overexpression restored the phosphorylation of downstream effectors (AKT and ERK1/2). Bioinformatics and dual‑luciferase reporter experiments identified miR‑218‑5p binding to the 3'-untranslated region of DBN1, which suppressed DBN1 expression. In addition, the experiments demonstrated that miR‑218‑5p acted as an upstream regulator of DBN1, and was involved in cell migration and invasion. Overall, DBN1 was upregulated in T‑ALL, and its depletion inhibited cell migration and invasion through downregulation of GAB2 and consequent inhibition of AKT and ERK signaling cascades. The present data suggested that DBN1 could be a novel biomarker of T‑ALL infiltration, which is a novel perspective in the field of leukemia research.
View Figures

Figure 1

DBN1 is highly expressed in T-ALL.
(A) DBN1 expression in BM cells from patients with T-ALL (n=170)
and healthy individuals (n=73) (GSE13159). 202806_at and
217025_s_at are the probe names of DBN1. Statistical significance
was determined using the Wilcoxon rank sum test. (B) DBN1
expression in T cells from healthy individuals (n=17), T-ALL cells
(n=15) and samples from patients with T-ALL (n=13) (GSE48558).
Statistical significance was determined using the Kruskal-Wallis
test followed by the Bonferroni post hoc test. (C) DBN1 expression
in samples from patients with T-ALL and healthy PBMCs in Oncomine
datasets (Haferlach leukemia and Haferlach leukemia 2). Statistical
significance was determined using the Wilcoxon rank sum test. (D)
DBN1 expression in the PBMCs from 10 patients with T-ALL and 6
healthy individuals. Statistical significance was determined using
an unpaired t-test. *P<0.05, ***P<0.001. BM, bone marrow;
DBN1, drebrin 1; PBMC, peripheral blood mononuclear cell; T-ALL,
T-cell acute lymphoblastic leukemia.

Figure 2

Knockdown of DBN1 inhibits cell
migration and invasion in vitro. (A) Reverse
transcription-quantitative PCR analysis of DBN1 mRNA expression in
DBN1-knockdown MOLT4 and Jurkat cells (sh-1 and sh-2). (B) Western
blot analysis of DBN1 protein expression in DBN1-knockdown MOLT4
and Jurkat cells (sh-1 and sh-2). The intensities of the protein
bands were determined using ImageJ. (C) Transwell assays showing
the migration tendency of DBN1-knockdown cells. (D)
Matrigel-Transwell assays showing the invasion tendency of leukemia
cells after DBN1 knockdown. The data were normalized and compared
with those in the sh-NC control group. Statistical significance was
determined using one-way ANOVA followed by the Bonferroni test.
**P<0.01, ***P<0.001. DBN1, drebrin 1; NC, negative control;
sh, short hairpin RNA.

Figure 3

RNA-seq analysis in DBN1-knockdown
MOLT4 cells. (A) Volcano plot of transcriptome profiling in MOLT4
shDBN1-1 cells vs. NC cells (left). Genes significantly upregulated
(red) or downregulated (green) following DBN1 silencing are
highlighted. Volcano plot for the MOLT4 shDBN1-2 group (right). (B)
Venn diagram of DEGs downstream of DBN1 in shDBN1-1 and shDBN1-2
cells. (C) Heatmap of 66 statistically significant DEGs between
DBN1 knockdown and control samples based on RNA-seq data. The
‘value’ represents the relative expression value (Z-score) obtained
after normalization. (D) Top 10 genes with the most significant
upregulation or downregulation in the shDBN1-1 group (left) and
shDBN1-2 group (right). Red and green bars represent upregulated
and downregulated DEGs, respectively. The color gradient reflects
significance levels, with darker shades denoting lower P-values.
DBN1, drebrin 1; DEG, differentially expressed gene; NC, negative
control; padj, adjusted P-value; RNA-seq, RNA sequencing; sh, short
hairpin RNA.

Figure 4

DBN1 regulates GAB2 expression. (A)
GAB2 FPKM in the NC and DBN1 knockdown groups. (B) RT-qPCR was
performed to assess the levels of GAB2 transcripts after DBN1
silencing. (C) Western blot analysis of GAB2 in NC and DBN1
knockdown cells. The intensities of the protein bands were
determined using ImageJ. (D) ANXA1 FPKM in the NC and DBN1
knockdown groups. (E) RT-qPCR was performed to determine the levels
of ANXA1 transcripts after DBN1 silencing. (F) Western blot
analysis of ANXA1 in NC and DBN1-knockdown cells. The intensities
of the protein bands were determined using ImageJ. Statistical
significance was determined using one-way ANOVA followed by the
Bonferroni test. *P<0.05, **P<0.01, ***P<0.001. ANXA1,
annexin A1; DBN1, drebrin 1; FPKM, fragments per kilobase million;
GAB2, Grb2-associated binding protein 2; NC, negative control; ns,
not significant; RT-qPCR, reverse transcription-quantitative PCR;
sh, short hairpin RNA.

Figure 5

GAB2 is highly expressed in T-ALL.
(A) GAB2 expression in the GSE13159 dataset. BM from patients with
T-ALL (n=170) and BM from healthy individuals (n=73). Statistical
significance was determined using the Wilcoxon rank sum test. (B)
GSE48558. T cells from healthy individuals (n=17), T-ALL cells
(n=15) and samples from patients with T-ALL (n=13). Statistical
significance was determined using the Kruskal-Wallis test followed
by the Bonferroni post hoc test. (C) Oncomine dataset (Haferlach
leukemia). Statistical significance was determined using the
Wilcoxon rank sum test. (D) GAB2 expression in 10 T-ALL clinical PB
samples and 6 healthy donor PB samples. Statistical significance
was determined using an unpaired t-test. (E) Spearman's correlation
between DBN1 and GAB2 expression based on healthy and T-ALL
samples. *P<0.05, ***P<0.001. BM, bone marrow; DBN1, drebrin
1; GAB2, Grb2-associated binding protein 2; PB, peripheral blood;
PBMC, peripheral blood mononuclear cell; T-ALL, T-cell acute
lymphoblastic leukemia.

Figure 6

Downregulation of DBN1 inhibits both
GAB2 expression and the activation of the PI3K/AKT/ERK pathways.
(A) Inhibitory effects on the expression levels of GAB2, and
phosphorylated AKT and ERK1/2 in the DBN1-knockdown MOLT4 cells.
(B) Jurkat cells. (C) Overexpression of GAB2 rescued the expression
levels of phosphorylated AKT and ERK1/2 in DBN1-knockdown MOLT4
cells. The intensities of the protein bands were determined using
ImageJ. Statistical significance was determined using one-way ANOVA
followed by the Bonferroni test. *P<0.05, **P<0.01,
***P<0.001. DBN1, drebrin 1; GAB2, Grb2-associated binding
protein 2; NC, negative control; ns, not significant; ox-,
overexpression; p-, phosphorylated; sh, short hairpin RNA.

Figure 7

miR-218-5p regulates DBN1 expression
through direct binding to the 3′-untranslated region of DBN1. (A)
Bioinformatics analysis was performed using the TargetScan, miRanda
and miRDB databases. (B) Predicted miR-218-5p binding sites in DBN1
(WT) and the designed mutant sequence (MUT). Relative luciferase
activity was determined after cells were transfected with WT or
MUT. Statistical significance was determined using an unpaired
t-test. (C) Reverse transcription-quantitative PCR demonstrated
miR-218-5p-mediated suppression of DBN1 expression in T-ALL cells.
Statistical significance was determined using an unpaired t-test.
(D) Western blotting demonstrated miR-218-5p-mediated suppression
of DBN1 expression in T-ALL cells. The intensities of the protein
bands were determined using ImageJ. Statistical significance was
determined using an unpaired t-test. (E) Expression levels of
miR-218-5p in samples from healthy individuals and T-ALL samples.
Statistical significance was determined using an unpaired t-test.
(F) Spearman's correlation between miR-218-5p and DBN1 expression
in healthy individuals and patients with T-ALL. (G) Spearman's
correlation between miR-218-5p and GAB2 expression in healthy
individuals and patients with T-ALL. **P<0.01, ***P<0.001.
CDS, coding sequence; DBN1, drebrin 1; GAB2, Grb2-associated
binding protein 2; LUC, luciferase; miR, microRNA; MUT, mutant; NC,
negative control; ns, not significant; T-ALL, T-cell acute
lymphoblastic leukemia; WT, wild-type.

Figure 8

miR-218-5p is involved in T-ALL cell
migration and invasion. (A) Effects of miR-218-5p mimics
transfection on T-ALL cell migration and invasion. Statistical
significance was determined by one-way ANOVA followed by the
Bonferroni test. (B) Western blot analysis of GAB2, and
phosphorylated and total AKT and ERK1/2 in the MOLT4 cells after
miR-218-5p mimic transfection. Statistical significance was
determined using an unpaired t-test. (C) Western blot analysis of
the effects of GAB2 overexpression on the levels of phosphorylated
AKT and ERK1/2 in MOLT4 cells transfected with miR-218-5p mimic.
The intensities of the protein bands were determined using ImageJ.
Statistical significance was determined using one-way ANOVA
followed by the Bonferroni test. *P<0.05, **P<0.01,
***P<0.001. DBN1, drebrin 1; GAB2, Grb2-associated binding
protein 2; miR, microRNA; NC, negative control; ns, not
significant; ox-, overexpression; p-, phosphorylated; T-ALL, T-cell
acute lymphoblastic leukemia.
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Copy and paste a formatted citation
Spandidos Publications style
Sun J, Huang X, Zeng X, Lei Y, He H, Wei Z, Xiao D, Zhang Q, Li X, Zhou F, Zhou F, et al: DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells. Oncol Rep 54: 149, 2025.
APA
Sun, J., Huang, X., Zeng, X., Lei, Y., He, H., Wei, Z. ... Shao, L. (2025). DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells. Oncology Reports, 54, 149. https://doi.org/10.3892/or.2025.8982
MLA
Sun, J., Huang, X., Zeng, X., Lei, Y., He, H., Wei, Z., Xiao, D., Zhang, Q., Li, X., Zhou, F., Shao, L."DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells". Oncology Reports 54.5 (2025): 149.
Chicago
Sun, J., Huang, X., Zeng, X., Lei, Y., He, H., Wei, Z., Xiao, D., Zhang, Q., Li, X., Zhou, F., Shao, L."DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells". Oncology Reports 54, no. 5 (2025): 149. https://doi.org/10.3892/or.2025.8982
Copy and paste a formatted citation
x
Spandidos Publications style
Sun J, Huang X, Zeng X, Lei Y, He H, Wei Z, Xiao D, Zhang Q, Li X, Zhou F, Zhou F, et al: DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells. Oncol Rep 54: 149, 2025.
APA
Sun, J., Huang, X., Zeng, X., Lei, Y., He, H., Wei, Z. ... Shao, L. (2025). DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells. Oncology Reports, 54, 149. https://doi.org/10.3892/or.2025.8982
MLA
Sun, J., Huang, X., Zeng, X., Lei, Y., He, H., Wei, Z., Xiao, D., Zhang, Q., Li, X., Zhou, F., Shao, L."DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells". Oncology Reports 54.5 (2025): 149.
Chicago
Sun, J., Huang, X., Zeng, X., Lei, Y., He, H., Wei, Z., Xiao, D., Zhang, Q., Li, X., Zhou, F., Shao, L."DBN1‑mediated upregulation of GAB2 facilitates the migration and invasion of T‑cell acute lymphoblastic leukemia cells". Oncology Reports 54, no. 5 (2025): 149. https://doi.org/10.3892/or.2025.8982
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