The synergistic effect of CDKN2B-AS1 and SPC25 on triple-negative breast cancer

Abstract

Background: Accumulating evidence suggests that long non-coding ribonucleic acid (RNA) cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) and messenger RNA (mRNA) spindle component 25 (SPC25) contribute to tumorigenesis and progression in various cancers. However, the synergistic effect between CDKN2B-AS1 and SPC25 has not yet been fully elucidated in triple-negative breast cancer (TNBC). This study sought to examine the synergistic effect of CDKN2B-AS1 and SPC25 and uncover a novel mechanism for the progression of TNBC.

Methods: The transcriptome profiles of TNBC in The Cancer Genome Atlas (TCGA) were calculated for differentially expressed genes (DEGs). Gene co-expression networks were constructed via a weighted correlation network analysis. We validated the relationship between CDKN2B-AS1 and SPC25 by bioinformatics and in-vitro studies (including Cell Counting Kit-8, transwell assays, and quantitative real-time polymerase chain reaction).

Results: CDKN2B-AS1 was found to be carcinogenic and was significantly upregulated and co-expressed with elevated SPC25 expression levels in the TNBC cells and sequencing profiles. Notably, the SPC25 mRNA levels were associated with poor clinical outcomes in TNBC patients. Specifically, the knockdown of CDKN2B-AS1 significantly inhibited TNBC cell proliferation and migration.

Conclusions: We identified a novel cancer-promoting regulation axis. The co-expression of CDKN2B-AS1 and SPC25 is expected to serve as a powerful candidate biomarker for diagnostic and prognostic purposes in TNBC.

Keywords: Triple-negative breast cancer (TNBC); cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1); proliferation; spindle component 25 (SPC25).

Figure 1

Identification of hub genes in WGCNA. (A,B) CDKN2B-AS1 and SPC25 expression was significantly higher in the TNBC tissues than the adjacent normal tissues. (C) The approximate soft threshold (power =7) was selected. (D) A genetic clustering tree-map was generated based on 56 gene modules using average-linkage hierarchical clustering. The modules were identified using the dynamic cut-tree method and named using various colors. (E) The heatmap analysis showed that the brown module exhibited the highest positive correlation with TNBC. (F) The interacting-relationship analysis of the co-expressed genes. The different colors in the vertical and horizontal axes represent different modules. The connection degree among different modules is indicated by the yellow intensity. (G) Scatter plot of module eigengenes in the brown module. (H) The Spearman correlation analysis showed that CDKN2B-AS1 and SPC25 were highly and positively correlated. (I-L) The GSEA analysis of genes in the brown module. WGCNA, weighted gene co-expression network analysis; CDKN2B-AS1, cyclin-dependent kinase inhibitor 2B antisense RNA 1; SPC25, spindle component 25; TNBC, triple-negative breast cancer; GSEA, gene set enrichment analysis.

Figure 2

Validation of SPC25 and functional annotation. (A,B) The mRNA expression of SPC25 and CDKN2B-AS1 is notably increased in most human cancer tissues compared to corresponding normal tissues. Red dots represent SPC25 expression in tumor samples. Green dots represent SPC25 expression in paired normal samples. (C) The upregulation of mRNA SPC25 was validated in GSE45827. (D) The protein expression of SPC25 in the CPTAC database was significantly higher in the TNBC samples than the normal samples. (E,F) SPC25 expression in normal breast tissues and BC tissues by immunohistochemical stain. Magnification, ×100. (G) According to the Human Protein Atlas database, SPC25 was localized in the cytoplasm of multiple human cells, such as U-2 OS and U-251 MG by immunofluorescence staining and photographed by confocal microscopy. Scale bar, 20 µm. (H) High SPC25 expression displayed a significant correlation with poor OS in TNBC patients. (I) SPC25-interaction proteins in TNBC. (J) A GO enrichment analysis was used to identify relevant the biological processes of the co-expressed genes. SPC25, spindle component 25; CDKN2B-AS1, cyclin-dependent kinase inhibitor 2B antisense RNA 1; CPTAC, Clinical Proteomic Tumor Analysis Consortium; OS, overall survival; TNBC, triple-negative breast cancer; BC, breast cancer; U-2 OS, human osteosarcoma cell line; U-251 MG, human glioblastoma cell line; GO, Gene Ontology.

Figure 3

The function of SPC25 in BC cells. (A,B) A single-cell analysis showed that SPC25 affected numerous cellular events, including cell-cycle regulation and DNA damage responses in BC. (C-F) SPC25 expression was positively correlated with above-mentioned biological processes. (G) Correlation between SPC25 and cell cycle–related genes. (H,I) RT-PCR validation of the relative expression of CDKN2B-AS1 and SPC25 in cell lines. (J) The relative expression of SPC25 was downregulated in si-CDKN2B-AS1–transfected MDA-MB-231 cells. (K) CCK-8 were performed to show that si-CDKN2B-AS1 inhibits the proliferation of MDA-MB-231. (L-N) Transwell were performed to detect the proliferation of MDA-MB-231 by crystal violet assay. (O) The flow cytometry assay showed that the apoptosis rate was significantly increased in CDKN2B-AS1 knockdown cells. *P<0.05, **P<0.01, ***P<0.001. ×20. SPC25, spindle component 25; BC, breast cancer; CDKN2B-AS1, cyclin-dependent kinase inhibitor 2B antisense RNA 1; CCK-8, cell counting kit-8.