Effect of miR-662 and miR-24-2-5p overexpressions on gene expression in human MDA-MB-231-luc2-NW1 (NW1) and MCF7 cell lines.

MicroRNAs (miRNAs), small non-coding RNAs, are powerful and extremely versatile regulators of the gene expression in cells, playing an important role in every step of cancer progression. Here, we report a list of transcripts regulated by miR-662 and miR-24-2-5p transient overexpressions using miRNA MIMIC transfection (MIMIC-miR-662 and MIMIC-miR-24-2-5p, respectively) compared to control (MIMIC-negCTRL-FAM+) at 36 hours post-transfection in human MDA-MB-231-luc2-NW1 (NW1) (for miR-662 and miR-24-2-5p) and MCF7 (only for miR-24-2-5p) breast cancer cell lines. We found 38 transcripts significantly deregulated (17 downregulated, 21 upregulated) in miR-662-overexpressing NW1 cells compared to mock-transfected cells; 222 (187 downregulated, 35 upregulated) and 34 (32 downregulated, 2 upregulated) transcripts deregulated in miR-24-2-5p-overexpressing NW1 and MCF7 cells, respectively, compared to mock-transfected cells. Furthermore, a total of 30 transcripts (29 downregulated, 1 upregulated) were shared between miR-24-2-5p-overexpressing NW1 and MCF7 cells compared to mock-transfected cells. For miR-662, which we found to promote breast cancer metastasis by stimulating cancer cell stemness (Puppo et al., 2023), we observed the deregulation of Cell Migration Inducing Hyaluronidase 1 (CEMIP), Interleukin 6 receptor (IL6R), High Mobility Group AT-Hook 2 (HMGA2), Small Ubiquitin Like Modifier 3 (SUMO3), and Polymerase (DNA) Delta Interacting Protein 2 (POLDIP2), which reinforced the evidence of the involvement of miR-662 in the acquisition of a stem-like phenotype by human NW1 breast cancer cells and miR-662 contribution to breast cancer bone metastasis formation. For miR-24-2-5p, we found that the downregulation of two transcripts, WD Repeat and FYVE Domain Containing 1(WDFY1) and SH3 Domain Binding Glutamate Rich Protein Like 2 (SH3BGRL2), might explain a possible role of miR-24-2-5p as a tumour suppressor by reducing breast cancer cell invasive properties. To investigate the functional role of two miRNAs, miR-662 and miR-24-2-5p, in the metastatic progression of breast cancer cells to distant organs, particularly to bone, we transiently overexpressed them in human breast cancer cell lines (MDA-MB-231-luc2-NW1 a.k.a. NW1, MCF7), and we then analysed whole transcriptomic changes at 36 hours post-transfection by RNA sequencing. MiR-662 and miR-24-2-5p transient overexpressions were obtained by miRNA MIMICs (50nM) transfection (MIMIC-miR-662 and MIMIC-miR-24-2-5p, respectively) using Lipofectamine 2000 as transfection agent. Three independent transfections (N=3) were performed for each condition. Gene expression profiling analysis was conducted by using data obtained by the RNA sequencing on two different cell lines (NW1, MCF7), where sample triplicates have been collected at 36 hours post-transfection. Each experimental condition (NW1/MIMIC-miR-662, NW1/MIMIC-miR-24-25p, MCF7/MIMIC-miR-24-2-5p) was compared to the relative control (NW1/MIMIC-negCTRL, MCF7/MIMIC-negCTRL).