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. 2023 Sep;13(9):e1427.
doi: 10.1002/ctm2.1427.

IGF2BP3 mediates the mRNA degradation of NF1 to promote triple-negative breast cancer progression via an m6A-dependent manner

Xu Zhang #  1 , Liang Shi #  1 , Han-Dong Sun #  1 , Zi-Wen Wang  1 , Feng Xu  1 , Ji-Fu Wei  2 , Qiang Ding  1
Affiliations

IGF2BP3 mediates the mRNA degradation of NF1 to promote triple-negative breast cancer progression via an m6A-dependent manner

Xu Zhang et al. Clin Transl Med. 2023 Sep.

Abstract

Background: N6-methyladenosine (m6A) is an abundant reversible modification in eukaryotic mRNAs. Emerging evidences indicate that m6A modification plays a vital role in tumourigenesis. As a crucial reader of m6A, IGF2BP3 usually mediates the stabilisation of mRNAs via an m6A-dependent manner. But the underlying mechanism of IGF2BP3 in the tumourigenesis of triple-negative breast cancer (TNBC) is unclear.

Methods: TCGA cohorts were analysed for IGF2BP3 expression and IGF2BP3 promoter methylation levels in different breast cancer subtypes. Colony formation, flow cytometry assays and subcutaneous xenograft were performed to identify the phenotype of IGF2BP3 in TNBC. RNA/RNA immunoprecipitation (RIP)/methylated RNA immunoprecipitation (MeRIP) sequencing and luciferase assays were used to certify the target of IGF2BP3 in TNBC cells.

Results: IGF2BP3 was highly expressed in TNBC cell lines and tissues. TET3-mediated IGF2BP3 promoter hypomethylation led to the upregulation of IGF2BP3. Knocking down IGF2BP3 markedly reduced the proliferation of TNBC in vitro and in vivo. Intersection co-assays revealed that IGF2BP3 decreased neurofibromin 1 (NF1) stabilisation via an m6A-dependent manner. NF1 knockdown could rescue the phenotypes of IGF2BP3 knockdown cells partially.

Conclusion: TET3-mediated IGF2BP3 accelerated the proliferation of TNBC by destabilising NF1 mRNA via an m6A-dependent manner. This suggests that IGF2BP3 could be a potential therapeutic target for TNBC.

Keywords: IGF2BP3; NF1; TET3; TNBC; m6A.

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Conflict of interest statement

The authors declare they have no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
IGF2BP3 was upregulated in triple‐negative breast cancer (TNBC) and correlated with a poor prognosis. (A) Heatmap showing RNA differential expression of eight N6‐methyladenosine (m6A) ‘readers’ between TNBC and non‐TNBC. (B) Expression of IGF2BP3 in normal tissue (n = 114) and breast cancer (n = 1097) from TCGA dataset. (C) Expression of IGF2BP3 in different molecular subtypes of breast cancer. (D) IGF2BP3 mRNA expression in 27 pairs of TNBC and adjacent normal tissues. The relative quantification was calculated by the 2−ΔΔCt method. (E) mRNA expression of IGF2BP3 in different cell lines. (F) Protein expression of IGF2BP3 in different cell lines. The relative quantification was calculated by the 2−ΔCt method. (G) Kaplan–Meier analysis of the overall survival of TNBC patients. Data are shown as the mean ± SEM; * p < .05.
FIGURE 2
FIGURE 2
The promoter of IGF2BP3 was hypomethylated in triple‐negative breast cancer (TNBC). (A) Schematic representation of the CpG islands in the IGF2BP3 promoter. The red region is the input sequence; the blue region is CpG islands. (B) Promoter methylation level of IGF2BP3 in different molecular subtypes of breast cancer tissues from TCGA dataset. (C) Methylation‐specific PCR of the CpG island of the IGF2BP3 promoter region in different breast cancer cell lines and matched normal breast cell line. (D and E) qRT‐PCR was used to confirm IGF2BP3 expression at the mRNA level. (F) Western blotting was used to confirm the IGF2BP3 and TET3 expression at the protein level. Data are shown as the mean ± SEM; * p < .05. (G) Methylation‐specific PCR of the CpG island of the IGF2BP3 promoter region in TET3 knockdown MDA‐MB‐231 and HCC‐1806 cells.
FIGURE 3
FIGURE 3
Knockdown of IGF2BP3 inhibited the proliferation and promoted the apoptosis of triple‐negative breast cancer (TNBC) in vivo and in vitro. (A and B) MDA‐MB‐231 and HCC‐1806 cell lines were transfected with lentivirus to knockdown IGF2BP3 expression (shIGF2BP3‐1, shIGF2BP3‐2). qRT‐PCR (A) and western blotting (B) were applied to verify the transfection efficiency. (C–H) CCK‐8, colony formation and EdU assays were performed in MDA‐MB‐231 and HCC‐1806 cell lines. (I and J) Flow cytometry assay and western blot were used to confirm the apoptosis analysis induced by the knockdown of IGF2BP3. (K–M) Tumour volume and weight in IGF2BP3 knockdown MDA‐MB‐231 cells compared with control at 4 weeks. Data are shown as the mean ± SEM; * p < .05.
FIGURE 4
FIGURE 4
Identification of the IGF2BP3 targets in triple‐negative breast cancer (TNBC). (A) Heatmap of differentially expressed genes (DEGs) performed by RNA sequencing. (B) GO enrichment analysis of DEGs. (C–F) GSEA plots show the pathways of IGF2BP3‐enriched DEGs. (G) N6‐methyladenosine (m6A) motif detection by DREME motif analysis and m6A sequencing results. (H) Percentage of different RNA species modified by m6A.
FIGURE 5
FIGURE 5
Neurofibromin 1 (NF1) was an N6‐methyladenosine (m6A) target of IGF2BP3 in triple‐negative breast cancer (TNBC). (A) Overlapping analysis of genes identified by m6A sequencing, RIP sequencing and RNA sequencing. (B) Distribution of m6A peaks and IGF2BP3‐binding peaks in transcripts. (C–E) Expression of NF1 was increased or decreased following IGF2BP3 knockdown or overexpression in MDA‐MB‐231 and HCC‐1806 cells at mRNA (C and D) and protein levels (E). Data are shown as the mean ± SEM; * p < .05. (F) Correlation analysis between IGF2BP3 and NF1 mRNA expression in TNBC tissues (n = 27). (G) Kaplan–Meier analysis of overall survival of TNBC patients.
FIGURE 6
FIGURE 6
IGF2BP3 regulated neurofibromin 1 (NF1) mRNA expression via N6‐methyladenosine (m6A)‐dependent manner. (A–D) MDA‐MB‐231 and HCC‐1806 cells were treated with 5 μg/mL actinomycin D (ActD) for 0, 2, 4 and 6 h, followed by qRT‐PCR and western blot analysis. (E–H) MDA‐MB‐231 and HCC‐1806 cell lysates were immunoprecipitated with IGF2BP3 or m6A antibody and control immunoglobulin G (IgG) to detect NF1 mRNA expression. (I) Schematic diagram of regions in the NF1 mRNA. (J and K) The luciferase activity for the reporter involving NF1‐A, B, C, D, E and pGL3 was transfected by knocking down IGF2BP3 in MDA‐MB‐231 and HCC‐1806 cells. (L) Schematic diagram of mutation regions in the NF1 mRNA. (M and N) The luciferase activity for the reporter involving NF1‐B, B‐mut, C and C‐mut was transfected in MDA‐MB‐231 and HCC‐1806 cells. Data are shown as the mean ± SEM; * p < .05.
FIGURE 7
FIGURE 7
Neurofibromin 1 (NF1) reversed the inhibition of proliferation and promotion of apoptosis induced by IGF2BP3 knockdown. (A and B) IGF2BP3 knockdown and the control groups of MDA‐MB‐231 and HCC‐1806 cells were transfected to knockdown NF1, confirmed by qRT‐PCR and western blotting. (C–F) CCK‐8 assays and colony formation assays were performed to analyse the proliferation ability of MDA‐MB‐231 and HCC‐1806 cells. (G and H) Flow cytometry assays was used to verify the apoptosis analysis of NF1 knockdown in shRNA‐NC and shIGF2BP3 cells. (I–K) Tumour volume and weight in NF1 knockdown and control groups in shRNA‐NC and shIGF2BP3 MDA‐MB‐231 cells in nude mice at different time points. Data are shown as the mean ± SEM; * p < .05.
FIGURE 8
FIGURE 8
Graphic illustration of IGF2BP3 modulating triple‐negative breast cancer (TNBC) proliferation and apoptosis via decreasing neurofibromin 1 (NF1) mRNA stability in an N6‐methyladenosine (m6A)‐dependent manner.
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