Downregulation of Sprouty homolog 2 by microRNA-21 inhibits proliferation, metastasis and invasion, however promotes the apoptosis of multiple myeloma cells

• Published in: Molecular medicine reports Vol. 12; no. 2; pp. 1810 - 1816
• Main Authors: WANG, JIN-HANG, ZHOU, WEN-WEN, CHENG, SHI-TONG, LIU, BO-XIN, LIU, FU-RONG, SONG, JIAN-QING
• Format: Journal Article
• Language: English
• Published: Greece D.A. Spandidos 01.08.2015; Spandidos Publications; Spandidos Publications UK Ltd
• Subjects: Apoptosis; Cancer therapies; Cell culture; Cell cycle; Cell growth; Cell Line, Tumor; Cell migration; Cell Movement; Cell Proliferation; Deoxyribonucleic acid; DNA; Down-Regulation; Flow cytometry; G1 phase; Gene expression; Gene regulation; Growth rate; Health aspects; Humans; Infections; Intracellular Signaling Peptides and Proteins - antagonists & inhibitors; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; invasion; Medical prognosis; Membrane Proteins - antagonists & inhibitors; Membrane Proteins - genetics; Membrane Proteins - metabolism; Metastases; Metastasis; MicroRNA; microRNA-21; MicroRNAs; MicroRNAs - antagonists & inhibitors; MicroRNAs - genetics; MicroRNAs - metabolism; miRNA; Multiple myeloma; Multiple Myeloma - metabolism; Multiple Myeloma - pathology; Multiplicity of infection; Oligonucleotides, Antisense - metabolism; Plasmids; Polymerase chain reaction; proliferation; Proteins; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering - metabolism; siRNA; sprouty homolog 2; Statistical analysis; Studies; Transfection; Tumors; Wound healing; China; United States > US; Germany
• ISSN: 1791-2997; 1791-3004
• DOI: 10.3892/mmr.2015.3567

The aim of the present study was to assess the effects of sprouty homolog 2 (SPRY2) gene regulation by miR-21 on the occurrence, development and tumor metastasis in multiple myeloma (MM). The miR-21 expression lentiviral vector (LV)-anti-miR-21 and a liposome transfection method were used to screen MM cell lines with stable silent SPRY2. Real-time quantitative polymerase chain reaction (PCR) and western blot analyses were used to detect SPRY2 expression and miR-21 protein expression levels. An MTT assay was used to assess cell proliferation. Flow cytometry was used for analysis of cell cycle. A scratch test/wound healing assay was used to detect the cell migration ability. A Transwell assay was used to detect the cell invasion ability. Real-time quantitative PCR and western blot analysis showed that in the MM cell lines with high endogenous miR-21 expression (RPMI8226 and KM3), SPRY2 expression was significantly lower. Conversely, in the U266 cell line with low endogenous miR-21 expression, SPRY2 expression was significantly higher, and the gray values of miR-21 and SPRY2 protein in the respective cell lines showed statistically significant differences (P<0.01). Following transfection of U266 cells, the expression of miR-21 in the U266/LV-anti-miR21 lentiviral multiplicity of infection (MOI) 20 group and -MOI 40 group decreased significantly compared with that in the untransfected U266 group (P<0.05). SPRY2 protein expression in U266 cells transfected with miR-21 mimics was significantly reduced compared with that in the non-transfected (untreated) group and the negative control-transfected group (P<0.01). An MTT assay showed that compared with the non-transfected and negative control groups, the cell growth rate as well as the proliferation rate were significantly decreased in the transfection group 48, 72 and 96 h after transfection (P<0.01). Flow cytometric analysis showed that 48 and 72 h after transfection of U266 cells with miR-21 mimics, the apoptotic rates were (24.7±1.97 and 38.6±1.56%) in the U266 group, (27.3±1.72 and 37.3±1.59%) in the siRNA group and (12.7±1.27 and 22.1±1.63%) in the U266/miR-21 group. Compared with the two control groups, the apoptotic rate in the U266/miR-21 group was significantly decreased and the G0/G1 phase cell population was significantly reduced (P<0.05). Scratch experiments showed that the cell migration ability was significantly reduced in the transfection group 24 and 48 h after transfection (P<0.05). A Transwell invasion assay confirmed that the number of U266 cells which migrated through a Matrigel-covered polyphosphate membrane significantly decreased in the transfection group 24 and 48 h after transfection. The cell-penetrating ability was also significantly decreased (P<0.05). In conclusion, the downregulation of SPRY2 gene expression mediated by miR-21 promotes the proliferation and invasion of MM cells in vitro, suggesting that miR-21 may be a novel potential molecular therapeutic target in the treatment of MM.