title: Downregulation of Sprouty homolog 2 by microRNA-21 inhibits proliferation, metastasis and invasion, however promotes the apoptosis of multiple myeloma cells.
abstract: 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.
authors.affiliation: Department of Laboratory Medicine, The First Hospital Affiliated to China Medical University Clinical Laboratory, Shenyang, Liaoning 110000, P.R. China.
authors.forename: Jin-Hang
authors.initials: JH
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine, The First Hospital Affiliated to China Medical University Clinical Laboratory, Shenyang, Liaoning 110000, P.R. China.
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Zhou
authors.affiliation: Department of Laboratory Medicine, The First Hospital Affiliated to China Medical University Clinical Laboratory, Shenyang, Liaoning 110000, P.R. China.
authors.forename: Shi-Tong
authors.initials: ST
authors.lastname: Cheng
authors.affiliation: Department of Laboratory Medicine, The First Hospital Affiliated to China Medical University Clinical Laboratory, Shenyang, Liaoning 110000, P.R. China.
authors.forename: Bo-Xin
authors.initials: BX
authors.lastname: Liu
authors.affiliation: Department of Cell Biology, China Medical University, Key Laboratory of Medical Cell Biology, Ministry of Public Health, Shenyang, Liaoning 110000, P.R. China.
authors.forename: Fu-Rong
authors.initials: FR
authors.lastname: Liu
authors.affiliation: Department of Laboratory Medicine, The First Hospital Affiliated to China Medical University Clinical Laboratory, Shenyang, Liaoning 110000, P.R. China.
authors.forename: Jian-Qing
authors.initials: JQ
authors.lastname: Song
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D015536:Down-Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C524940:MIRN21 microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; C485561:SPRY2 protein, human
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