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

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.

Correlation between microRNA­21 and sprouty homolog 2 gene expression in multiple myeloma.

The aim of the present study was to investigate the expression level of microRNA 21 (miR­21) in the peripheral blood of patients with multiple myeloma (MM) and to investigate the correlation between miR­21 and sprouty homolog 2 (SPRY2) gene expression levels in MM. A total of 30 patients with MM, 15 with monoclonal gammopathy of undetermined significance (MGUS) and 20 normal control (NC) outpatients were selected for the detection of miR­21 and SPRY2 expression using reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis was performed to detect the expression of miR­21 and SPRY2 in MM cell lines. The expression of miR­21 in U­266 cells following lipofectamine transfection of fluorescence­labeled miR­21 mimic/inhibitor was observed using a fluorescence microscope and the expression level of SPRY2 in the miR­21 mimic/inhibitor­transfected U­266 cells was detected using western blot analysis. The miR­21 expression level in the circulating serum of the MM patient group was significantly higher (P<0.01) than that of the MGUS and NC groups. The MM cell lines with high endogenous miR­21 expression exhibited an expression level of SPRY2 that was significantly lower than that in the MM cells with low endogenous miR­21 expression. The transfection efficiency of fluorescence­labeled miR­21 mimic/inhibitor was >90%. Compared with the miR­21 expression level in untreated U­266 cells (0.82±0.13), the expression level of miR­21 was increased by 120.2­fold in miR­21 mimic­transfected cells (98.6±14.2; P<0.001) and was decreased by 61.9% in the miR­21 inhibitor­transfected cells (0.37±0.06; P<0.05). The grayscale value of protein bands demonstrated that SPRY2 protein expression significantly decreased in miR­21 mimic­transfected U­266 cells compared with that in the inhibitor­transfected, siRNA­transfected and untreated cells (P<0.01). miR­21 may represent a negative regulator involved in the downregulation of SPRY2 in MM. miR­21 is closely associated with the pathogenesis, progression and prognosis of MM and may thus be used as an indicator of poor MM prognosis.