The lncRNA GATA3-AS1/miR-495-3p/CENPU axis predicts poor prognosis of breast cancer via the PLK1 signaling pathway

Abstract

The function of centromere protein U (CENPU) gene in breast cancer has not been well understood. Therefore, we explored the expression profiles of CENPU gene in breast carcinoma to better understand the functions of this gene, as well as the relationship between CENPU expression and the prognosis of breast carcinoma patients. Our results indicate that CENPU was expressed at significantly higher levels in cancerous tissues than in normal tissues. Furthermore, CENPU expression correlated significantly with many clinicopathological characteristics of breast cancer. In addition, we discovered that high levels of CENPU expression predicted poor prognosis in patients with breast cancer. Functional investigation revealed that 180 genes exhibited co-expression with CENPU. Functional annotation indicated that 17 of these genes were involved in the PLK1 signaling pathway, with most of them (16/17) being expressed at significantly higher levels in malignant tissues compared with normal controls and correlating with a poor prognosis. Subsequently, we found that four miRNAs, namely hsa-miR-543, hsa-miR-495-3p, hsa-miR-485-3p, and hsa-miR-337-3p, could be regarded as potential CENPU expression regulators. Then, five lncRNAs were predicted to potentially bind to the four miRNAs. Combination of the results from expression, survival, correlation analysis and functional experiments analysis demonstrated the link between lncRNA GATA3-AS1/miR-495-3p/CENPU axis and prognosis of breast cancer. In conclusion, CENPU could be involved in cell cycle progression through PLK1 signaling pathway.

Keywords: CENPU; bioinformatics analysis; breast cancer; lncRNA; miRNA.

Figure 1

CENPU expression in normal and cancerous tissues. (A) CENPU mRNA expression in different normal tissues from the HPA database. (B) CENPU mRNA expression in different cancer tissues from the HPA database. (C–D) The mRNA and protein level expression of CENPU in breast cancer compared with normal tissue by analyzing UALCAN database, ^**P < 0.01.

Figure 2

Differences in CENPU expression between breast cancer patients with different clinicopathological features. The clinicopathological features analyzed included (A) age, (B) SBR grade, (C) NPI score, (D) nodal status, (E) ER status, (F) PR status, (G) HER2 status, (H) HU's subtype, (I) RSSPC subtype, (J) basal-like status, (K) triple-negative status, (L) triple-negative and basal-like status.

Figure 3

Determination of prognostic significance of CENPU expression (Affymetrix IDs are valid: 218883_s_at and 229305_at) in patients with breast cancer from Kaplan-Meier plotter database. (A, D) OS curves established according to CENPU expression. (B, E) RFS curves established according to CENPU expression. (C, F) DMFS curves established according to CENPU expression.

Figure 4

Identification and functional analysis of genes that exhibited co-expression with CENPU. (A) The Venn diagram of CENPU’s co-expressed genes from UALCAN, GEPIA, and cBioPortal databases. (B–D) GO functional annotation (biological process, cellular component, and molecular function) for 180 co-expressed genes of CENPU. (E–G) Pathway (NCI-Nature, Reactome, and KEGG) enrichment analysis for these 180 co-expressed genes of CENPU.

Figure 5

(A–Q) Prognostic value of the 17 genes that exhibited co-expression with CENPU and are involved in the PLK1 pathway in breast cancer. ^**P < 0.01.

Figure 6

Gene set enrichment analyses were performed using gene expression data of 226 African-American women with triple negative breast cancer (TNBC). (A) Genes are enriched in PLK1 pathway. (B) Genes are enriched in Fisher G2-M cell cycle. (C) Genes are enriched in the Reactome cell cycle mitotic. (D) Genes are enriched in Reactome cell cycle. False discovery rate (FDR) q value, normalized enrichment score (NES), and P values are shown, vs: versus.

Figure 7

(A–G) Prognostic significance of the seven miRNAs that can potentially regulate CENPU expression in breast cancer analyzed by Kaplan-Meier plotter database.

Figure 8

Expression and biological significance of the lncRNA GATA3-AS1 in breast carcinoma. (A) Expression levels of GATA3-AS1 in breast carcinoma as determined with GEPIA. (B) Expression levels of GATA3-AS1 in breast cancer as determined with the starBase database. (C) Prognostic value of GATA3-AS1 in breast cancer as determined with GEPIA. (D–F) Relative expression levels of GATA3-AS1, CENPU, and miR-495-3p in MDA-MB-468, BT-549, HCC1954, MCF-7, and MCF-10A cell lines as determined by qRT-PCR. (G) Western blotting analysis of CENPU protein levels in MCF-7 cells after the cells were transfected with sh-NC, sh-GATA3-AS1#1, sh-GATA3-AS1#2. (H) Quantitative RT-PCR analysis of miR-495-3p levels in MCF-7 cells after the cells were transfected with sh-NC, sh-GATA3-AS1#1, sh-GATA3-AS1#2. (I) Proliferation of MCF-7 cells after the cells were transfected with sh-NC, sh-GATA3-AS1#1, sh-GATA3-AS1#2 by MTT assay. (J) The identified lncRNA-miRNA-mRNA axis that can predict the prognosis of breast cancer patients. ^**P < 0.01, ^*P < 0.05.