Identification by Comprehensive Bioinformatics Analysis of KIF15 as a Candidate Risk Gene for Triple-Negative Breast Cancer

Abstract

Background: Previous studies have shown that kinesin family proteins (KIFs) play an indispensable roles in several types of cancer. However, the expression and clinical significance of KIFs in triple-negative breast cancer remain unclear.

Methods: In this study, the role of KIF15, including gene expression analysis, methylation characteristic, CNV characteristic, and miRNA target regulation, was evaluated using multiple bioinformatic tools based on TCGA database. Quantitative real-time PCR and Western blot were used to determine the expression level of KIF15 in triple-negative breast cancer cell lines. Then, functional experiments were employed to explore the effects of KIF15 on tumor growth and metastasis in triple-negative breast cancer.

Results: Our data showed that KIF15 was significantly upregulated in triple-negative breast cancer (TNBC). Functionally, downregulation of KIF15 significantly facilitated apoptosis and G2/M phase arrest, and inhibited the migration and invasion of TNBC cells. The mechanism of action of KIF15 was closely related to DNA replication checkpoint and cell cycle regulation in TNBC based on GSEA. In addition, bioinformatics analysis demonstrated that high expression of KIF15 in TNBC was correlated with copy number aberration and DNA methylation levels.

Conclusion: Our findings suggest that KIF15 is a novel oncogene in TNBC and provide us a strong evidence that it might be served as a potential clinical target and biomarker in triple-negative breast cancer.

Keywords: bioinformatics analysis; biomarker; kinesin family proteins; triple-negative breast cancer.

Figure 1

The mRNA level of KIF15 in various types of human pan-cancer (GEPIA). (A). KIF15 is upregulated in 29 and downregulated in 1 type of cancer. Red and the green bar graphs refer to tumour (T) and normal tissues (N), respectively. (B). The mRNA level of KIF15 in human TNBC and normal tissues.

Figure 2

The effects of KIF15 knockdown on cell cycle and apoptosis in MDA-MB-231 cells (A) Transcriptional expression of KIF15 in various triple-negative breast cancer cell lines. (B). Western blot images (left) and quantification (right) of KIF15 at the protein level in various triple-negative breast cancer cell lines. (C). KIF15-knockdown MDA-MB-231 stable cell lines, and qPCR confirming the knockdown. (D). Western blot images (left) and quantifications (right) of for the efficiency test of KIF15-knockdown MDA-MB-231 stable cell lines. (E). Detection of apoptosis of MDA-MB-231 cells by Annexin V/PI double staining. (F). Detection of the cell cycle of MDA-MB-231 cells by PI staining. **P <0.01. ***P <0.001.

Figure 3

KIF15-related upstream mechanisms. (A) Five online tools were used to predict miRNAs targeting KIF15. The correlations between KIF15 and has-miR-135a-5p (B), hsa-miR-139-5p (C), hsa-miR-381-3p (D), hsa-miR-664a-5p (E), hsa-miR-2115-5p (F) in breast cancer were analyzed . Copy number change (G) and methylation level (H) and of KIF15 is associated with mRNA expression.

Figure 4

The effects of KIF15 knockdown on the migration and invasion in MDA-MB-231 cells. (A). The effects of KIF15 knockdown on migration (images on the left and quantification on the right) in MDA-MB-231 cells. (B). The effects of KIF15 knockdown on invasion (images on the left and quantification on the right) in MDA-MB-231 cells. (C). The protein–protein interaction network in breast cancer. (D). Two pathways (DNA damage and cell cycle) are associated with KIF15 in breast cancer according to GSEA. FDR is the false discovery rate, and the NES is the normalized enrichment score. ***P <0.001.