miR-126 Decreases Proliferation and Mammosphere Formation of MCF-7 and Predicts Prognosis of ER+ Breast Cancer

Abstract

Breast cancer (BC) is a major health burden that affects over one million women each year. It is the most prevalent cancer in women and the number one cancer killer of them worldwide. Of all BC subtypes, estrogen receptor-positive (ER+) BC is the most commonly diagnosed. The objective of this study is to investigate the contribution of miR-126 in the tumorigenesis of ER+ BC. miR-126 was downregulated in ER+ BC tissues from young breast cancer patients, as shown through miRNA microarray analysis and RT-qPCR. Subsequently, the effect of the modulation of miR-126 levels on the proliferation, cell cycle progression, and spheres formation of the ER+ BC cell line, MCF-7, was assessed by MTT assay, PI analysis, and mammosphere formation assay, respectively. miR-126 overexpression significantly decreased MCF-7 proliferation and mammosphere-forming ability, but did not affect cell cycle progression. Then, in silico analysis determined SLC7A5, PLXNB2, CRK, PLK2, SPRED1, and IRS1 as potential targets of miR-126. RT-qPCR data showed that miR-126 overexpression significantly downregulated SLC7A5 and PLXNB2 mRNA levels in MCF-7. Finally, in silico survival analysis showed that high expression of miR-126 or low expression of SLC7A5 correlated with better overall survival (OS) of ER+ BC patients. Overall, our study suggests that miR-126 might play a tumor suppressor role in ER+ BC. miR-126 and SLC7A5 might also be considered potential prognostic biomarkers in ER+ BC.

Keywords: SLC7A5 (LAT1); breast cancer; estrogen receptor-positive; miR-126.

Figure 1

miR-126 expression in ER+ BC tissues versus NAT. (A) Heatmap of the differentially expressed miRNA in breast tumor tissue samples versus NAT taken from Lebanese ER+ BC patients < 40 years old (fold change > 2 and adjusted p-value < 0.05). Each row represents a differentially expressed miRNA and each column represents one of the 24 samples. The color key shown in the top left illustrates the relative expression level of an miRNA across all samples. (B) Dot plots show the fold change of miR-126 in 40 BC tissues normalized to the average of 19 NAT with RNU6B used as an endogenous control. Error bars represent SEM. *** denotes p < 0.001.

Figure 2

Transfection efficiency of miR-126 mimic in MCF-7. Percentage of transfection in FAM-labeled NC and miR-126 mimic–transfected cells as compared to untransfected control (CTL). Error bars represent SEM (n = 3). *** denotes p < 0.001.

Figure 3

Effect of miR-126 on cell proliferation and cell cycle progression. (A): Proliferation of miR-126 mimic compared to NC-transfected cells by MTT assay at 48 and 72 h post transfection (n = 3). (B): Cell Cycle Analysis of the transfected cells by PI staining at 24 h (n = 3) and 48 h (n = 3) post transfection. (C): Representative figure of the flow cytometric analysis of the cell cycle 24 h post transfection. Error bars represent SEM. * denotes p < 0.05.

Figure 4

Mammosphere-forming ability of miR-126 mimic compared to NC-transfected cells. (A): Representative images of mammospheres formed by NC- and miR-126-transfected MCF-7. Scale bar: 100 µm. (B): Mammosphere-forming efficiency (MFE) of transfected MCF-7. Error bars represent SEM (n = 4). * denotes p < 0.05.

Figure 5

Expression levels of potential miR-126 targets in miR-126 mimic compared to NC-transfected MCF-7 24 h post transfection by RT-qPCR. GAPDH was used as an internal control. Error bars represent SEM (n = 3). * denotes p < 0.05.

Figure 6

Correlation of the expression of miR-126 or SLC7A5 with overall survival (OS) of ER+ BC patients. In silico Kaplan–Meier plot of (A) hsa-miR-126 expression with OS of 944 ER+ BC patients, (B) SLC7A5 expression with OS of 720 ER+ BC patients. HR: hazard ratio.