Prognostic role of elevated mir-24-3p in breast cancer and its association with the metastatic process

Abstract

MicroRNAs have been shown to play important roles in breast cancer progression and can serve as biomarkers. To assess the prognostic role of a panel of miRNAs in breast cancer, we collected plasma prospectively at the time of initial diagnosis from 1,780 patients with stage I-III breast cancer prior to definitive treatment. We identified plasma from 115 patients who subsequently developed distant metastases and 115 patients without metastatic disease. Both groups were matched by: age at blood collection, year of blood collection, breast cancer subtype, and stage. The median follow up was 3.4 years (range, 1-9 years). We extracted RNA from plasma and analyzed the expression of 800 miRNAs using Nanostring technology. We then assessed the expression of miRNAs in primary and metastatic breast cancer samples from The Cancer Genome Atlas (TCGA). We found that, miR-24-3p was upregulated in patients with metastases, both in plasma and in breast cancer tissues. Patients whose primary tumors expressed high levels of miR-24-3p had a significantly lower survival rate compared to patients with low mir-24-3p levels in the TCGA cohort (n=1,024). RNA-Seq data of the samples with the highest miR-24-3p expression versus those with the lowest miR-24-3p in the TCGA cohort identified a specific gene expression signature for those tumors with high miR-24-3p. Possible target genes for miR-24-3p were predicted based on gene expression and binding site, and their effects on cancer pathways were evaluated. Cancer, breast cancer and proteoglycans were the top three pathways affected by miR-24-3p overexpression.

Keywords: breast cancer; gene expression profiling.

Figure 1

(A) Volcano plot of differentially expressed miRNAs in metastatic and non-metastatic serum samples obtained by Nanostring data analysis. Upregulated miRNAs are plotted in red and downregulated in blue (B) Volcano plot of differentially expressed miRNAs in TCGA stage I versus stage IV primary tumor samples. Upregulated miRNAs in Stage IV are plotted in red and downregulated in blue (C) Survival curve of mir-24-3p in TCGA patients (D). Distribution of mir-24-3p across different tumor subtypes in TCGA primary tumors.

Figure 2

(A) Box-plot of mir-24-3p levels in patients showing the highest (n=25) and lowest (n=25) expressing levels of mir-24-3p in primary tumors from the TCGA (B) t-SNE plot of patients with high and low mir-24-3p expressing levels showing the separation of the two conditions (C) Volcano plot of the genes differentially expressed based on mir-24-3p expression in primary tumors (high versus low) (D) Heatmap of the top 500 genes differentially expressed in patients with high versus low mir-24-3p levels.

Figure 3

(A) The top three significantly (P value < 0.05) effected pathways in patients with high mir-24-3p vs. low mir-24-3p (n=50) (B) Heat map overview of the principal pathways involved in cancer (KEGG: hsa05200). Upregulated genes are in red and downregulated genes are shown in green.

Figure 4

Circos plots depicting (A) 316 deferentially expressed genes (FDR < 0.1) out of 745 target genes detected by TargetScan for mir-24-3p (differentially expressed in patients with high mir-24-3p levels vs. low miR-24). Red arcs show genes upregulated and blue downregulated in samples with high mir-24-3p and the histogram represents the fold change. (B) 27 significantly down regulated genes (FDR < 0.1 & fold change > 2) out of 745 target genes detected by TargetScan for mir-24-3p showing the most possible targets.