GRB2 Promotes Malignant Behaviors of Breast Cancer by Modulating the Global Expression and Alternative Splicing Profiles in SK-BR-3 Cells Through Binding mRNA

Abstract

Purpose: The flexible protein GRB2 interacts with HER1-4 on the cell surface and regulates the development of tumor cells; meanwhile, it is also an RBP that plays an important role in post-transcriptional regulation in eukaryotes, which affects every stage of mRNA synthesis, modification, splicing, and stabilization. Although some studies have found a connection between GRB2 and HER2-overexpression breast cancer, highlighting the potential of GRB2 as a novel biomarker that stimulates tumor growth, limited data were available to elaborate on their interaction mechanisms.

Methods: In this research, we found 396 different gene expressions between the Grb2-knockdown group and the SK-BR-3 group by the RNA sequencing approach. After GRB2 was knocked down, 956 alternative splicing events occurred.

Results: The fRIP-seq results showed that GRB2-binding reads were significantly enriched in the intron region, indicating that UUAGC and UUGGUUGG might be the binding motifs. An integration analysis of DEGs with the peak genes of fRIP-seq revealed that 63 genes possess GRB2 binding sites on their mRNAs or antisense RNAs. By integration analysis of AS events with the peak genes of fRIP-seq, 66 genes related to AS events were found.

Conclusions: Above, these AS events may be regulated by GRB2 to promote the progression of HER2-overexpression breast cancer.

Keywords: GRB2; RBPs; alternative splicing; breast cancer.

FIGURE 1

GRB2 is elevated and has a poor prognosis in breast cancer patients. (A) Box plot shows the mRNA of GRB2 in normal and tumor tissues from UALCAN. (B) Box plot shows the protein level of GRB2 in normal and tumor tissues. (C) GRB2 immunohistochemical demonstration from the HPA database. (D) The survival curve for GRB2 in breast cancer patients from UALCAN. BRCA, breast invasion carcinoma.

FIGURE 2

GRB2 knockdown in cells. (A) RT‐PCR shows expression of mRNA in cells. (B) Western blot demonstrates the effectiveness of the GRB2 knockdown. SI (1–3): GRB2 knockdown by small interfering RNA; NC (1–3): Control group.

FIGURE 3

GRB2 regulates gene expression in cells. (A) PCA of samples after normalizing gene expression levels. (B) Volcano plot showing DEGs between si_GRB_2 and si_NC samples. (C) Heat map shows the level of DEGs. (D) Scatter plot displays the GO:BP of up‐regulated genes. (E) Scatter plot displays the GO:BP of down‐regulated genes. (F) Bar plot displays the pattern and difference of DEGs. ***p < 0.001.

FIGURE 4

GRB2 regulates gene alternative splicing in SK‐BR‐3 cells. (A) Bar plot shows all RASEs in samples. (B) Boxplot shows the ratio of ASEs in samples. (C) Bar plot shows the gene number of ASEs. (D) Scatter plot exhibits the enriched GO:BP of the RASGs. (E) Bar plot shows the expression pattern and difference of genes. A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; ASEs, alternative splicing events; MXE, mutually exclusive exons; RASEs, regulated alternative splicing events; RASGs, regulated alternative splicing genes. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

GRB2 binds to mRNAs in cells. (A) Western blot of GRB2 immunoprecipitates (IP) via an anti‐Flag monoclonal antibody. (B) Pie chart illustrates the genomic distribution of GRB2‐bound peaks. (C) Motif results reveal the presence of enriched motifs in the GRB2‐bound peaks. (D) Venn diagram illustrates the overlapped peaks. (E) Scatter plot exhibits the most enriched GO:BP results.

FIGURE 6

Integration exploration between GRB2‐bound RNAs and DEGs. (A) Venn diagram shows the overlapped genes between DEG and peaks genes. (B) Heat map shows the level of overlapped genes. (C) Bar plot shows the pattern and difference of DEGs.(D) GRB2‐binding peak genes of MESD. (E) GRB2‐binding peak genes of DIP2C. ***p < 0.001.

FIGURE 7

Integration exploration between GRB2‐bound RNAs and RASGs. (A) Venn diagram shows the overlapped peaks. (B) Scatter plot exhibits the enriched GO:BP results of peak genes. (C) Bar plot shows the expression pattern and difference of AS genes. (D) GRB2 regulates AS of GLS. (E) GRB2 modulates the process of AS for FBOX22. *p < 0.05; **p < 0.01.