Differences of time-dependent microRNA expressions in breast cancer cells

Abstract

MicroRNA (miRNA) expression is a dynamic process in the cell, and the proper time period for post-transcriptional regulation might be critical due to the gene-on/-off expression times of the cell. Here, we investigated the effect of different time-points on proliferation, invasion and miRNA expression profiles of human breast cancer cell lines MCF-7 (non-metastatic, epithelium-like breast cancer cell line with oestrogen receptor (ER) positive (+) and human breast cancer cell lines MDA-MB-435 (metastatic, invasive, ER negative (-). For this purpose, MCF-7 and MDA-MB-435 cells were seeded different number in E-plate 16 for proliferation experiment using an electrical impedance-based real-time cell analyzer system (RTCA) for 168 h. Similarly, invasion potential of MCF-7 and MDA-MB-435 were determined by RTCA for 90 h. Total RNAs including miRNAs were isolated at 2, 4, 6, 12, 24, 48 h from the MCF-7 and MDA-MB-435 cells. Afterward, the quantitative 84 miRNA expressions of MCF-7 and MDA-MB-435 were analyzed by Fluidigm Microfluidic 96.96 Dynamic Array. The results of these study demonstrated that both proliferation potential and invasion capacity of MDA-MB-435 is higher than MCF-7 as time-dependent manner. Furthermore, we detected that up/down expressions of 32 miRNAs at all time points in MDA-MB-435 compared to MCF-7 (at least ten-fold increased). Because of the high number of miRNAs, we more closely evaluated the expression of six of them (miR-100-5p, miR-29a-3p, miR-130a-3p, miR-10a-5p, miR-10b-5p, miR-203a), and determined that their levels were dramatically changed by at least 50-fold at different time points of the experiment (p < 0.01). The expression levels of five of these miRNAs (miR-100-5p, miR-10a-5p, miR-10b-5p, miR-130a-3p, and miR-29a-3p) started to increase from the fourth hour and continued to increase until the 48th hour in MDA-MB-435 cells compared to MCF-7 cells (p < 0.01). Simultaneously, the expression of one of these miRNAs (miR-203a) decreased from the sixth hour to the 48th hour in MDA-MB-435 as compared to MCF-7. We determined pathways associated with target genes using mirPath - DIANA TOOLS. Small RNAs including miRNA are essential regulatory molecules for gene expressions. In the literature, gene expressions have been published as burst and pulse in the form of discontinuous transcription. The data of the research suggested that time-dependent changes of miRNA expressions can be affected target gene transcriptional fluctuations in breast cancer cell and can be base for the further studies.

Keywords: Discontinuous transcription; MCF-7; MDA-MB-435; miRNA.

Fig. 1

Real time analysis of proliferation graph of MCF-7 and MDA-MB-435 cells using E-plate 16. Notes: The MCF-7 and MDA-MB-435 cells seeded to 10.000 and 20.000 cell per well into the E-plate-16 (n = 8), respectively. As shown in the graphs, the aggressiveness of different cell number of MDA-MB-435 (10.000 cell per well- turquoise curve; 20.000 cell per well-pink curve) was higher than different cell number of MCF-7 (10.000 cell per well-green curve; 20.000 cell per well-blue curve) (p < 0.05).

Fig. 2

Comparison of MCF-7 and MDA-MB-435 cell invasion capacities generated by real-time cell analyzer measuring impedance-based signals. MCF-7 ( 20.000 cell per well; green curve) and MDA-MB-435 (20.000 cell per well; green curve) were seeded in a 1:20 (v/v) Matrigel-coated CIM-Plate 16 with 10% serum serving as the chemoattractant in the lower chamber. The rate of invasion was monitored in real-time using the RTCA system. Comparison of cell index between the MCF-7 cells (green curve) and MDA-MB-435 cells (blue curve) invading the matrigel layer towards 10% FBS DMEM, during 90 h (p < 0.05, n = 6).

Fig. 3

Photographs of polycarbonate filters coated with Matrigel from transwell-plate chemoinvasion assays. Invaded MCF-7 cells in 1:10 matrigel, 1:20 matrigel and invaded MDA-MB-435 cells in 1:10 matrigel, 1:20 matrigel.

Fig. 4

Dramatically expressed (p<0.01) of six miRNAs in MDA-MB-435 cells compared to MCF-7 cells for different time points (2, 4, 6, 12, 24, 48h).

Fig. 5

The numbers of target genes regulated by these 6 dramatically expressed miRNAs (p<0.05) in pathways as analyzed by mirPath-DIANA TOOLS.