MiR-21下调SPRY2表达对多发性骨髓瘤细胞增殖和侵袭能力的影响及其机制研究

The influence of miR- 21 reduce SPRY2 expression in multiple myeloma cell proliferation on the invasive ability and proliferation and its mechanism

目的:探讨miR-21下调SPRY2基因表达在多发性骨髓瘤(multiple myeloma,MM)发生发展以及转移过程中的作用。方法:构建miR-21表达载体LV-anti-miR-21及脂质体转染法筛选稳定沉默SPRY2的MM细胞系。应用实时定量PCR和Western blot检测miR-21表达和SPRY2蛋白表达水平。四甲基偶氮唑蓝法(MTT法)检测细胞增殖能力;流式细胞仪分析细胞周期;划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力。结果:实时定量PCR及Western blot检测结果表明:转染U266细胞后,U266/LV-anti-miR-21慢病毒MOI 20组和MOI 40组miR-21表达量较U266/un组差异明显下降(P<0.05);转染miR-21 mimics组的U266细胞SPRY2的蛋白表达量较无转染组(untreated)和阴性对照组(siRNA)明显降低,差异均具有统计学意义(P<0.01)。与无转染组、阴性对照组相比:MTT法显示转染组48、72、96h细胞的生长速度明显减慢,增殖率明显下降(P<0.01);流式细胞术表明miR-21 mimics转染U266细胞48、72h后,细胞凋亡率分别为U266组[(24.7±1.97)%、(38.6±1.56)%]、siRNA组[(27.3±1.72)%、(37.3±1.59)%]和U266/miR-21组[(12.7±1.27)%、(22.1±1.63)%],与两对照组比较,U266/miR-21组细胞凋亡率明显降低,G0/G1期细胞明显减少,差异有统计学意义(P<0.05)。划痕实验显示转染组24、48h细胞迁移的能力明显减弱(P<0.05);Transwell实验证实转染组48、72h细胞穿过聚磷酸酯膜的U266细胞数明显减少,细胞穿膜能力明显降低(P<0.05)。结论:MiR-21下调SPRY2基因表达能够在体外条件下促进MM细胞的增殖和侵袭作用,揭示其在MM的发生、发展及转移过程中的作用及可能的机制,为确立新分子靶向治疗提供可靠的研究依据。

Objective：To discuss the miR-21 reducing SPRY2 gene expression in the process of development and transfer of multiple myeloma.Methods：To build miR-21 expression vector LV-anti-miR-21 and liposome transfection method screening stable silence SPRY2 MM cell line.Real-time quantitative PCR and Western blot were used to detect miR-21 expression and the expression level of SPRY2 protein.Methyl thiazolyl tetrazolium（MTT） detect the ability of cell proliferation ; Flow cytometry instrument analysis the cell cycle.Wound healing detect cell migration.Transwell test detect cell invasive ability.Results：Real-time quantitative PCR and Western blot tests showed that：After transfection U266 cells,U266/LV-anti-miR-21 lentivirus MOI 20 groups and MOI 40 groups miR-21 expression quantity was obviously lower than U266/un group（P ＜ 0.05）.The protein expression level of transfection miR-21 mimics SPRY2 U266 cells was obviously lower than that without transfection group（untreated） and the negative control group,the differences were statistically significant（P ＜ 0.01）.Compared with no transfection group and negative control group,MTT method showed transfection group 48,72,96 hours cell growth were slowly significantly,proliferation rate significantly decreased（P ＜0.0 1）.Flow cytometry showed that miR-21 mimics transfection U266 cells 48,72 hours,the apoptosis rate of U266 group were respectively [（24.7-± 1.97） ％,（38.6 ±-1.56） ％],siRNA group[（27.3±1.72）％,（37.3 ±1.59）％] and U266/miR-21 group[（12.7±1.27）％,（22.1 ±1.63）％],compared with two controls,U266 cell miR-21 group apoptosis rate was obviously lower,G0/G1 phase cells decreased significantly,the difference was statistically significant（P ＜ 0.05）.Wound healing showed transfection group cell migration ability decreased significantly in 24,48h（P ＜ 0.05）.Transwell test confirm cells through the poly phosphate film U266 cells was significantly reduced in 48,72h of transfection group,cell penetrating membrane ability was decreased obviously（P ＜ 0.05）.Conclusion：MiR-21 reduce SPRY2 gene expression and promote multiple myeloma cells in vitro proliferation and invasion in vitro,of multiple myeloma and process,provide reliable basis to establish new molecular targeted therapy.