Mechanism of IGF2BP2/CCNB2 axis promoting the progression of triple negative breast cancer

doi:10.3877/cma.j.issn.1674-0807.2025.05.005

Abstract

Abstract:

Objectives

To investigate the role and mechanism of the m6A-dependent IGF2BP2/CCNB2 axis in the progression of triple negative breast cancer (TNBC).

Methods

Tumor and adjacent normal tissue specimens were collected from 30 TNBC patients who underwent surgery in the Department of Breast and Thyroid Surgery, the First Affiliated Hospital of Army Medical University between May 2020 and April 2023. Human normal breast epithelial cell line MCF-10A and TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-549, HCC1806, and MCF-10A) were used. Real-time quantitative RT-PCR was used to measure IGF2BP2 mRNA between tumor tissue and adjacent normal tissue, and among different cell lines. The MDA-MB-231 cells were transfected with the IGF2BP2 overexpression plasmid (OE-IGF2BP2 group), IGF2BP2 siRNA sequence (si-IGF2BP2 group), wild-type CCNB2 reporter plasmid pmirGLO-CCNB2-WT and empty vector OE-NC (WT+NC group), wild-type CCNB2 reporter plasmid pmirGLO-CCNB2-WT and IGF2BP2 overexpression plasmid OE-IGF2BP2 (WT+OE group), mutant-type CCNB2 reporter plasmid pmirGLO-CCNB2-Mut and empty vector OE-NC (Mut+NC group), mutant-type CCNB2 reporter plasmid pmirGLO-CCNB2-Mut and IGF2BP2 overexpression plasmid OE-IGF2BP2 (Mut+OE group), IGF2BP2 overexpression plasmid and CCNB2 siRNA (OE+si-CCNB2 group). Real-time quantitative RT-PCR was used to measure IGF2BP2 mRNA expression in each group. Cell proliferation assays were used to measure optical density; Transwell assays were used to assess cell migration and invasion; dual-luciferase reporter were used to detect the ratio of luciferase activity of wild-type and mutant m6A sites on CCNB2 mRNA. The methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) were used to validate the binding activity between IGF2BP2 and CCNB2. The continuous data were compared between two groups using t tests; among multiple groups using one-way ANOVA and pairwise comparisons by Tukey's test. The mRNA expression levels of two factors between tumor tissue and adjacent tissues were compared by paired t test; multiple-group comparisons at different time points were analyzed by repeated measures analysis of variance with pairwise comparison by Tukey's test. The correlation between IGF2BP2 and CCNB2 mRNA expression was analyzed by Spearman method.

Results

IGF2BP2 mRNA expression in adjacent normal tissue was significantly higher than that in TNBC tumor tissue (1.58±0.59 vs 6.51±1.83, t=15.620, P<0.001). The IGF2BP2 mRNA expression in patients with regional lymph node metastasis (n=19) was significantly higher than that in those without (n=11) (4.62±1.03 vs 7.61±1.16, t=7.075, P<0.001). The IGF2BP2 mRNA expression in stage Ⅲ–Ⅳ breast cancer patients (n=22) was significantly higher than that in stage Ⅰ–Ⅱ breast cancer patients (n=8) (7.34±1.31 vs 4.24±0.77, t=6.261, P<0.001). The IGF2BP2 mRNA expression in MCF-10A and TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-549, HCC1806) was 1.09±0.11, 4.34±0.20, 2.25±0.17, 1.67±0.16, and 3.18±0.09, respectively, indicating a significant difference (F=281.70, P<0.001). The IGF2BP2 mRNA expression was significantly higher in OE-IGF2BP2 group than in control group (3.32±0.18 vs 1.05±0.06, t=23.610, P<0.001); lower in si-IGF2BP2 group than in control group (0.33±0.04 vs 0.66±0.05, F=81.310, P<0.001). The optical density values in control, OE-IGF2BP2 and si-IGF2BP2 groups showed a significant difference (between groups: F=300.00, P<0.001; time points: F=947.20, P<0.001; interaction: F=45.390, P<0.001); migration cell counts were 186.30±8.08, 254.70±6.03, 122.70±8.62, respectively, indicating a significant difference (F=222.80, P<0.001); invasion cell counts were 152.70±11.02, 222.70±9.45, 81.67±7.02, respectively, indicating a significant difference (F=172.10, P<0.0001); CCNB2 mRNA expression was 1.05±0.07, 2.95±0.07, 0.62±0.04, respectively, indicating a significant difference (F=1193, P<0.001); CCNB2 mRNA stability differed significantly among groups (between groups: F=72.430, P<0.001; time points: F=511.30, P<0.001; interaction: F=23.930, P<0.001). CCNB2 mRNA expression in TNBC tissue was significantly higher than that in adjacent tissue (5.03±1.62 vs 1.42±0.49, t=13.140, P<0.001), and CCNB2 mRNA expression was positively correlated with IGF2BP2 mRNA (r=0.7043, P<0.001). The ratio of luciferase activity in WT+OE group were significantly higher than that in control group (12.33±1.11 vs 5.33±0.79, t=8.895, P<0.001). The methylated CCNB2 enrichment was significantly increased in the anti-m6A group (t=15.94, P<0.001), and CCNB2 enrichment was also elevated in the anti-IGF2BP2 group (t=19.61, P<0.001). The optical density values in control, OE+si-NC, and OE+si-CCNB2 groups showed a significant difference (between groups: F=122.40, P<0.001; time points: F=2279.000, P<0.001; interaction: F=24.530, P<0.001); migration cell counts were 144.00±4.58, 258.00±5.29, and 175.00±8.89, respectively, indicating a significant difference (F=244.300, P<0.001); invasion cell counts were 113.30±11.50, 214.70±7.37, and 152.70±9.71, respectively, indicating a significant difference (F=83.590, P<0.001).

Conclusion

IGF2BP2 enhances the stability of CCNB2 mRNA in an m6A-dependent manner, thus promoting proliferation, migration, and invasion of TNBC cells.

Key words: Breast neoplasms, Triple negative breast cancer, RNA-binding proteins, Gene expression regulation，neoplastic

Qingqiu Chen, Ling Zhong, Ting Zhang, kongyong Zhang, Yu Gui, Xiaowei Qi, Lin Ren. Mechanism of IGF2BP2/CCNB2 axis promoting the progression of triple negative breast cancer[J]. Chinese Journal of Breast Disease(Electronic Edition), 2025, 19(05): 287-295.

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URL: https://zhrxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-0807.2025.05.005

https://zhrxbzz.cma-cmc.com.cn/EN/Y2025/V19/I05/287

Figures/Tables 7

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引物名称	引物序列
IGF2BP2	F： 5’-CTACGCCTTCGTGGACTACC-3’
	R： 5’-CATCCAACACCTCCCACTG-3’
CCNB2	F： 5’-CTCGGAGAGCAGTCCTAACG-3’
	R： 5’-CAAATCACTGGACACCGTCG-3’
GAPDH	F： 5’-TGACTTCAACAGCGACACCCA-3’
	R： 5’-CACCCTGTTGCTGTAGCCAAA-3’

引物名称	引物序列
IGF2BP2	F： 5’-CTACGCCTTCGTGGACTACC-3’
	R： 5’-CATCCAACACCTCCCACTG-3’
CCNB2	F： 5’-CTCGGAGAGCAGTCCTAACG-3’
	R： 5’-CAAATCACTGGACACCGTCG-3’
GAPDH	F： 5’-TGACTTCAACAGCGACACCCA-3’
	R： 5’-CACCCTGTTGCTGTAGCCAAA-3’

组别	0 h	24 h	48 h	72 h	96 h
NC组	0.27±0.04	0.53±0.04	1.08±0.09	1.53±0.03	1.81±0.02
OE-IGF2BP2组	0.27±0.06	0.58±0.03	1.58±0.05^a	1.89±0.07^a	2.23±0.10^a
si-IGF2BP2组	0.27±0.09	0.49±0.02^b	0.64±0.05^ab	1.03±0.08^ab	1.32±0.06^ab
F值	0.006	5.672	138.716	133.382	128.761
P值	0.994	0.041	<0.001	<0.001	<0.001

组别	0 h	24 h	48 h	72 h	96 h
NC组	0.27±0.04	0.53±0.04	1.08±0.09	1.53±0.03	1.81±0.02
OE-IGF2BP2组	0.27±0.06	0.58±0.03	1.58±0.05^a	1.89±0.07^a	2.23±0.10^a
si-IGF2BP2组	0.27±0.09	0.49±0.02^b	0.64±0.05^ab	1.03±0.08^ab	1.32±0.06^ab
F值	0.006	5.672	138.716	133.382	128.761
P值	0.994	0.041	<0.001	<0.001	<0.001

组别	0 h	3 h	6 h	9 h	12 h
NC组	1.09±0.35	0.74±0.04	0.67±0.02	0.57±0.05	0.40±0.04
OE-IGF2BP2组	1.03±0.07	0.92±0.03^a	0.81±0.02^a	0.72±0.04^a	0.66±0.03^a
si-IGF2BP2组	1.06±0.06	0.62±0.03^ab	0.47±0.05^ab	0.37±0.03^ab	0.23±0.03^ab
F值	0.807	70.391	81.295	56.086	137.187
P值	0.489	<0.001	<0.001	<0.001	<0.001

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