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. 2021 Jan-Dec:20:15330338211019506.
doi: 10.1177/15330338211019506.

Investigation of Candidate Genes and Pathways in Basal/TNBC Patients by Integrated Analysis

Qi Liu  1   2   3 , Xiang Song  2 , Zhaoyun Liu  1   2 , Zhiyong Yu  2
Affiliations
Free PMC article

Investigation of Candidate Genes and Pathways in Basal/TNBC Patients by Integrated Analysis

Qi Liu et al. Technol Cancer Res Treat. 2021 Jan-Dec.
Free PMC article

Abstract

Purpose: This study aims to identify the key pathway and related genes and to further explore the potential molecular mechanisms of triple negative breast cancer (TNBC).

Methods: The transcriptome data and clinical information of breast cancer patients were downloaded from the TCGA database, including 94 cases of paracancerous tissue, 225 cases of Basal like type, 151 cases of Her2 type, 318 cases of Luminal type A, 281 cases of Luminal type B, and 89 cases of Normal Like type. The differentially expressed genes (DEGs) were identified based on the criteria of |logFC|≥1.5 and adjust P < 0.001.Their functions were annotated by gene ontology (GO) analysis and Kyoto Encyclopedia of differentially expressed genes & Genomes (KEGG) pathway analysis. Cox regression univariate analysis and Kaplan-Meier survival curves (Log-rank method) were used for survival analysis. FOXD1, DLL3 and LY6D were silenced in breast cancer cell lines, and cell viability was assessed by CCK-8 assay. Further, the expression of FOXD1, DLL3 and LY6D were explored by immunohistochemistry on triple negative breast tumor tissue and normal breast tissue.

Results: A total of 533 DEGs were identified. Functional annotation showed that DEGs were significantly enriched in intermediate filament cytoskeleton, DNA-binding transcription activator activity, epidermis development, and Neuroactive ligand-receptor interaction. Survival analysis found that FOXD1, DLL3, and LY6D showed significant correlation with the prognosis of patients with the Basal-like type (P < 0.05). CCK-8 assay showed that compared with Doxorubicin alone group, the cytotoxicity of Doxorubicin combined with siRNA-knockdown of FOXD1, DLL3, or LY6D was much significant.

Conclusion: The DEGs and their enriched functions and pathways identified in this study contribute to the understanding of the molecular mechanisms of TNBC. In addition, FOXD1, DLL3, and LY6D may be defined as the prognostic markers and potential therapeutic targets for TNBC patients.

Keywords: The Cancer Genome Atlas; identification of key genes; survival prognosis; triple negative breast cancer.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
A, Flow chart of this study. B, Venn diagram of aberrant expression profiles of mRNAs between Basal like vs. Normal, Basal like vs. Her2, Basal like vs. Luminal A, Basal like vs. Luminal B, Basal like vs. Normal like. A total of 533 intersecting mRNAs were identified.
Figure 2.
Figure 2.
Volcano plot used to display the differentially expressed genes screened in each group. (A) Normal vs. Basal like, (C) Her2 vs. Basal like, (E) Luminal A vs. Basal like, (G) Luminal B vs. Basal like, (I) Normal like vs. Basal like. Heatmap visualization of a subset of the top 50 up- and downregulated DEGs across different groups. (B) Normal vs. Basal like, (D) Her2 vs. Basal like, (F) Luminal A vs. Basal like, (H) Luminal B vs. Basal like, (J) Normal like vs. Basal like.
Figure 3.
Figure 3.
GO terms analysis of DEGs using cluster profiler. Cnetplot used to show the potential biological complexity of the same gene that may belong to multiple annotation categories. (A-B) Cellular component (CC), (C-D) molecular function (MF), (E-F) biological process (BP). (G) KEGG pathway enrichment analysis of DEGs.
Figure 4.
Figure 4.
(A) The PPI network visualizated by Cytoscape for the DEGs. Red and blue circles represent up-regulated and down-regulated genes, respectively. Top 3 modules from the protein-protein interaction network. (B) Module 1, (C) module 2, (D) module 3.
Figure 5.
Figure 5.
Prognostic value of 3 genes—FOXD1 (A), DLL3 (B), LY6D (C)—in Basal-like cancer patients.
Figure 6.
Figure 6.
Cytotoxicity of doxorubicin for breast cancer cell lines. Cell viability was assessed by CCK-8 assay, each group was repeated for 3 time. The data of group was analyzed by T test, value of P < 0.05 was significant statistical differences between the groups. The error bars represent SD. These 3 cell lines show different sensitivity to DOX (A). BT549 cells were treated with 0.5 μM DOX alone or combined with si-FOXD1 respectively, DMSO acted as the control, and then subjected to CCK8 assay (B). BT549 cells were treated with 0.5 μM DOX alone or combined with si-DLL3 respectively, DMSO acted as the control, and then subjected to CCK8 assay (C). MDA-MB-468 cells were treated with 0.5 μM DOX alone or combined with si-LY6D respectively, DMSO acted as the control, and then subjected to CCK8 assay (D).
Figure 7.
Figure 7.
The transfection efficiency of si-FOXD1, si-DLL3 and si-LY6D were verified by RT-PCR.
Figure 8.
Figure 8.
Expression of DLL3, FOXD1, and LY6D (SP×100 and SP×400) in TNBC tissue: Expression of DLL3 in TNBC tissue (SP×100 and SP×400) (A, B). Expression of FOXD1 in TNBC tissue (SP×100 and SP×400) (D, E). Expression of LY6D in TNBC tissue (SP×100 and SP×400) (G, H). Expression of DLL3, FOXD1, and LY6D (SP×100) in normal breast tissue (C, F, I).
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