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 miR21 expression lentiviral vector (LV)antimiR21 and a liposome transfection method were
used to screen MM cell lines with stable silent SPRY2. Realtime quantitative polymerase chain reaction (PCR) and western blot analyses were used to detect SPRY2 expression and miR21 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. Realtime quantitative PCR and western blot analysis showed that in the MM cell lines with high endogenous miR21 expression (RPMI8226
and KM3), SPRY2 expression was significantly lower. Conversely, in the U266 cell line with low endogenous miR21 expression, SPRY2 expression was significantly higher, and the gray values of miR21 and SPRY2 protein in the respective cell lines showed statistically significant differences (P<0.01).
Following transfection of U266 cells, the expression of miR21 in the U266/LVantimiR21 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 miR21 mimics was significantly reduced compared with that in the nontransfected (untreated) group and the negative controltransfected group (P<0.01). An MTT assay showed that compared with the nontransfected 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 miR21 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/miR21 group. Compared with the two control groups, the apoptotic rate in the U266/miR21 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 Matrigelcovered polyphosphate membrane significantly decreased in the transfection group 24 and 48 h after transfection. The cellpenetrating 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.
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Document Type: Research Article
Department of Laboratory Medicine, The First Hospital Affiliated to China Medical University Clinical Laboratory, Shenyang, Liaoning 110000, P.R. China
Department of Cell Biology, China Medical University, Key Laboratory of Medical Cell Biology, Ministry of Public Health, Shenyang, Liaoning 110000, P.R. China
Publication date: August 1, 2015
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Molecular Medicine Reports is a monthly, peer-reviewed journal available in print and online, that includes studies devoted to molecular medicine, underscoring aspects including pharmacology, pathology, genetics, neurosciences, infectious diseases, molecular cardiology and molecular surgery. In vitro and in vivo studies of experimental model systems pertaining to the mechanisms of a variety of diseases offer researchers the necessary tools and knowledge with which to aid the diagnosis and treatment of human diseases.
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