Bozkurt Serife Buket, Ozturk Bahadir, Kocak Nadir, Unlu Ali
University of Selcuk, Faculty of Medicine, Department of Medical Biochemistry, Konya, Turkey.
Hacettepe University, Research Center of Dental Faculty, Ankara, Turkey.
Noncoding RNA Res. 2020 Dec 19;6(1):15-22. doi: 10.1016/j.ncrna.2020.12.001. eCollection 2021 Mar.
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.
微小RNA（miRNA）表达是细胞中的一个动态过程，由于细胞的基因开启/关闭表达时间，转录后调控的合适时间段可能至关重要。在此，我们研究了不同时间点对人乳腺癌细胞系MCF-7（非转移性、上皮样乳腺癌细胞系，雌激素受体（ER）阳性（+））和人乳腺癌细胞系MDA-MB-435（转移性、侵袭性、ER阴性（-））增殖、侵袭及miRNA表达谱的影响。为此，将不同数量的MCF-7和MDA-MB-435细胞接种于E-plate 16中，使用基于电阻抗的实时细胞分析仪系统（RTCA）进行168小时的增殖实验。同样，使用RTCA测定MCF-7和MDA-MB-435的侵袭潜能，时长为90小时。在2、4、6、12、24、48小时从MCF-7和MDA-MB-435细胞中分离包括miRNA在内的总RNA。随后，通过Fluidigm微流控96.96动态阵列分析MCF-7和MDA-MB-435的84种miRNA定量表达。这些研究结果表明，MDA-MB-435的增殖潜能和侵袭能力均高于MCF-7，且呈时间依赖性。此外，我们检测到与MCF-7相比，MDA-MB-435在所有时间点均有32种miRNA的上调/下调表达（至少增加10倍）。由于miRNA数量众多，我们更深入地评估了其中6种（miR-100-5p、miR-29a-3p、miR-130a-3p、miR-10a-5p、miR-10b-�p、miR-203a）的表达，并确定在实验的不同时间点它们的水平至少有50倍的显著变化（p < 0.01）。与MCF-7细胞相比，MDA-MB-435细胞中这5种miRNA（miR-100-5p、miR-10a-5p、miR-10b-5p、miR-130a-3p和miR-29a-3p）的表达水平从第4小时开始升高，并持续升高至第48小时（p < 0.01）。同时，与MCF-7相比，MDA-MB-435中这些miRNA之一（miR-203a）的表达从第6小时至第48小时下降。我们使用mirPath - DIANA TOOLS确定了与靶基因相关的途径。包括miRNA在内的小RNA是基因表达的重要调控分子。在文献中，基因表达已以不连续转录的爆发和脉冲形式发表。该研究数据表明，miRNA表达的时间依赖性变化可能影响乳腺癌细胞中的靶基因转录波动，并可为进一步研究提供依据。
