多发性骨髓瘤细胞中c-myc介导的CKS1B转录调控的研究

摘要

目的 以人多发性骨髓瘤细胞株XG1、XG7为研究对象,探讨原癌基因c-myc介导的CKS1B转录调控机制. 方法 构建c-myc表达质粒和CKS1B启动子报告质粒,转染多发性骨髓瘤细胞XG1及XG7,应用荧光素酶报告基因试验检测c-myc对CKS1B启动子活性的影响；再采用RNA干扰技术构建c-myc-shRNA载体,转染XG1及XG7细胞株,Western免疫印迹测定CKS1B蛋白表达水平的改变. 结果 荧光素酶报告基因试验显示,对照组相对荧光素酶活性为0.046±0.052,XG1-CKS1Bpro组为0.159±0.067,XG1 -CKS1 Bpro+ c-myc组为0.656±0.086,3组间存在统计学差异(P＜0.05).XG7-CKS1 Bpro组相对荧光素酶活性为0.162±0.081,XG7-CKS1 Bpro+ c-myc组为0.663±0.077,3组间存在统计学差异(P＜0.05).在XG1、XG7细胞内c-myc均可转录激活CKS1B启动子活性.2组细胞株中,shRNA干扰沉默c-myc基因表达后CKS1B蛋白水平均明显下调. 结论 c-myc介导了CKS1B基因转录调控,c-myc在多发性骨髓瘤的发病与预后中可能具有重要作用.%Objective To investigate the effect of c-myc on SKC1B transcription in XG1 and XG7 multiple myeloma cells. Methods The c-myc expression plasmid and the CKS1B reporter plasmid were constructed. XG1 cells were transiently co-transfected with c-myc expression plasmid and CKS1B reporter plasmid, c-myc-mediated CKS1B transcriptional activity was assessed using luciferase reporter gene assay; The c-myc-mediated transcriptional activity was confirmed by silencing c-myc expression through shRNA-mediated RNA interference. CKS1B expression was verified by western blotting. Results In reporter gene assays, the relative luciferase activity in the control group was 0.046 ±0. 052. When co-transfected with c-myc expression plasmid and CKS1B reporter plasmid, the relative luciferase activity was 0. 159 ±0.067 and 0.656 ±0. 086 respectively in XG1 cells( P < 0. 05); and was 0. 162 ± 0. 081 and 0.663 ± 0. 077 respectively in XG7 cells, (P<0.05). Thus, c-myc overexpression gave rise to the increased activity of CKS1B promotor. Once the c-myc gene expression was silenced by shRNA interference, the protein level of CKS1B were significantly decreased in both XG1 and XG7 cells. Conclusions These results suggest c-myc may activate CKS1B expression, and play a critical role in the diagnosis and prognosis of multiple myeloma.