microRNA expression profiling on individual breast cancer patients identifies novel panel of circulating microRNA for early detection

Abstract

Breast cancer (BC) is the most common cancer type and the second cause of cancer-related death among women. Therefore, better understanding of breast cancer tumor biology and the identification of novel biomarkers is essential for the early diagnosis and for better disease stratification and management choices. Herein we developed a novel approach which relies on the isolation of circulating microRNAs through an enrichment step using speed-vacuum concentration which resulted in 5-fold increase in microRNA abundance. Global miRNA microarray expression profiling performed on individual samples from 23 BC and 9 normals identified 18 up-regulated miRNAs in BC patients (p(corr) < 0.05). Nine miRNAs (hsa-miR-4270, hsa-miR-1225-5p, hsa-miR-188-5p, hsa-miR-1202, hsa-miR-4281, hsa-miR-1207-5p, hsa-miR-642b-3p, hsa-miR-1290, and hsa-miR-3141) were subsequently validated using qRT-PCR in a cohort of 46 BC and 14 controls. The expression of those microRNAs was overall higher in patients with stage I, II, and III, compared to stage IV, with potential utilization for early detection. The expression of this microRNA panel was slightly higher in the HER2 and TN compared to patients with luminal subtype. Therefore, we developed a novel approach which led to the identification of a novel microRNA panel which was upregulated in BC patients with potential utilization in disease diagnosis and stratification.

Figure 1. Heatmap depicting the expression of 18 circulating miRNAs in patients with breast cancer (BC) compared to normal healthy controls.

Heatmap of individual plasma samples of 9 control and 23 patients with BC. The miRNA expression levels exhibited ≥2.0 fold changes and p ≤ 0.05 are presented. Each column represents an individual sample and each row represents a single miRNA. Expression level of each miRNA in a single sample is depicted according to the color scale.

Figure 2. Validation of the expression of 9 miRNAs identified from microarray data employing plasma or serum from patients with breast cancer (BC, n = 46) and healthy controls (n = 14) using quantitative Real-Time PCR.

P values were calculated using unpaired t-test and are indicated on each plot. Data are presented as “−delta CT” using box and whiskers plots.

Figure 3. Expression of 9 circulating miRNAs according to breast cancer stage.

The expression of a group of 9 circulating miRNAs measured using qRT-PCR in normal (n = 14) or patients with breast cancer (BC, n = 46) are plotted as a function of cancer stage. P values were calculated using unpaired t-test. Data are presented as “−delta CT” using box and whiskers plots. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, n.s., not significant.

Figure 4. Expression of 9 circulating miRNAs according to breast cancer molecular subtype.

The expression of a group of 9 circulating miRNAs measured using qRT-PCR in normal (n = 14) or patients with breast cancer (BC, n = 46) are plotted as a function of BC molecular type. P values were calculated using unpaired t-test. Data are presented as “−delta CT” using box and whiskers plots. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, n.s., not significant, TN: triple negative, HER2: HER2+.