GRK6对多发性骨髓瘤MM1R细胞增殖的作用及其作用机制的初步研究

Effect of GRK6 on Proliferation of Multiple Myeloma MM1R Cells and Its Mechanism

目的:探讨GRK6对多发性骨髓瘤细胞增殖作用的调控及其机制。方法:通过构建干扰人GRK6基因的慢病毒载体GRK6-shRNA,转染筛选获得稳定下调GRK6基因表达的多发性骨髓瘤细胞株MM1R。利用实时定量PCR及Westem blot验证慢病毒载体介导的GRK6基因表达下调的效果。选取GRK6表达下调最显著的细胞株检测GRK6对细胞增殖的影响。结果:构建干扰人GRK6基因的慢病毒载体GRK6-shRNA,转染多发性骨髓瘤MM1R细胞,筛选并获得稳定下调GRK6基因的多发性骨髓瘤细胞株MM1R。CCK-8试验结果显示,实验组细胞增殖活性明显低于对照组(P<0.05)。流式细胞术检测显示:实验组细胞在G0/G1期被阻滞(P<0.05)。Western Blot检测实验组Cyclin D1和CDK4水平相比较对照组明显降低。结论:成功构建了特异性干扰GRK6表达的慢病毒载体,获得可稳定下调GRK6的MM1R细胞株并且发现下调GRK6表达后,MM1R细胞可能通过抑制Cyclin D1和CDK4水平使MM1R细胞阻滞于G0/G1期,并显著地抑制了多发性骨髓瘤MM1R细胞的增殖。

Objective：To explore the regulatory effect of GRK6 on the proliferation of multiple myeloma cells and its mechanism.Methods：A lentivirus vector shRNA interfering in human GRK6 gene expression was constructed and transfected into multiple myeloma cells to obtain the cell line MMIR with stable down-regulation of GRK6 gene expression.The real-time quantitative PCR and Western blot were used to confirm the effectiveness of the GRK6 gene expression down-regulation mediated by lentivirus vector.The MMIR cells with most obvious down-regulation were selected to detect the effect of GRK6 gene on cell proliferation.Results：The lentivirus vector GRK6-shRNA interfering in human GRK6 gene was constructed succesufully and transfected into multiple myeloma cells,thereby the MMIR cell line with stable down-regulation of GRK6 gene was obtained.The CCK-8 assay showed that the proliferative viability of MMIR cells in experimental group was significantly lower than that in control group（P〈0.05）;the flow cytometry showed that cells in experimental group were arrested in G0/G1 phase（P〈0.05）;the Western blot detection showed that the Cyclin D1 and CDK4 levels in experiment group obviously decreased as compared with control group.Conclusion：A lentivirus vector which can specifically interfere in GRK6 gene expression is constructed successfully,The MMIR cell line with stable down-regulation of GRK6 expression is obtained by transfection and screening.The down-regulation of GRK6 expression can arrest MMIR cells in G0/G1 phase,moreover inhibits the proliferation of MMIR cells by inhibition of Cyclin D1 and CDK4 levels.