Experimental study on the proliferation and invasion activity of the c-maf gene inhibited multiple myeloma RPMI8226 cells
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摘要:
目的 探讨c-maf基因对多发性骨髓瘤细胞增殖、侵袭力的影响。 方法 脂质体法用c-maf siRNA转染多发性骨髓瘤 细胞系RPMI8226，RT-PCR技术检测c-maf基因的表达，MTT实验和Transwell小室分别检测多发性骨髓瘤细胞增殖活性和侵袭 力的改变，流式细胞术检测细胞周期，Western blots检测相关蛋白的表达情况，并检测Caspase的活性。 结果 c-maf siRNA有效地 转染入细胞并抑制了c-maf基因的表达。转染c-maf siRNA细胞的增殖活性及体外侵袭力均显著下降（P＜0.05），细胞周期阻滞在 G2/M 期，survivin、MMP-2、MMP-9、ARK5、cyclin B1 蛋白水平明显低于对照组（P＜0.05），Caspase-3/7 蛋白活性高于对照组（P＜ 0.05）。 结论 c-maf siRNA可明显抑制多发性骨髓瘤RPMI8226细胞的增殖活性及侵袭力。c-maf基因可做为多发性骨髓瘤基因治疗的靶基因。 Abstract:Objective To investigate the effect of c-maf gene on the MM cells ' proliferation and invasion activity. Methods Lipofectin Reagent was used to transfect c-maf siRNA into multiple myeloma cell of RPMI8226. The mRNA expression level of c-maf was detected by RT-PCR.Cell growth curve was measured by MTT assay. Transwell chamber test was used to measure MM cells ' in vitro invasion activity. The cell cycle distribution were assessed by flow cytomentry. The protein expression levels of survivin，MMP-2, MMP-9, ARK5 and cyclin B1 were detected by Western blot. We also detected the activity of Caspase-3/7. Results The c-maf siRNA was effectively transfected into cells and the mRNA expression of the c-maf gene was inhibited.MTT test and Transwell chamber test showed that the proliferation and in vitro invasion activity of transfected cells were significantly lower than those of other two groups (P＜0.05). Cell cycle of c-maf siRNA transfected group cells was arrested in G2/M phase. The expression levels of survivin，MMP-2，MMP-9，ARK5，cyclin B1 and the activity of Caspase-3/7 between c-maf siRNA transfected group and the other two groups were statistically different (P＜0.05). Conclusion c-maf gene by c-maf siRNA can remarkably inhibit proliferation and invasion of multiple myeloma cell lines of RPMI8226. C-maf gene may be used as the target for multiple myeloma gene therapy. -
Key words:
- c-maf gene /
- multiple myeloma /
- RNAi /
- proliferation /
- invasion, apoptosis
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图 1 转染24 h后倒置荧光显微镜下观察转染后实验细胞（×100）
Figure 1. MM cells under the fluoroscope at 24 h after transfectedion（×100）
图 2 c-maf siRNA对多发性骨髓瘤细胞系RPMI8226 c-maf基因表达的影响
Figure 2. Effect of siRNA on c-maf mRNA expression in RPMI8226 cells
图 3 转染c-maf siRNA对人多发性骨髓瘤细胞系RPMI8226增殖的影响
Figure 3. The growth curve according to the MTT assay in different groups of RPMI8226 after transfection with c-maf siRNA
图 4 c-maf基因沉默对人多发性骨髓瘤细胞系RPMI8226体外侵袭力的影响（*P＜0.05）
Figure 4. The effect of c-maf siRNA on in vitro invasion activity of RPMI8226 cells
图 5 c-maf基因沉默对人多发性骨髓瘤细胞系RPMI8226细胞周期的影响
Figure 5. The effect of c-maf siRNA on cell cycle progression of RPMI8226
A：0 h；B：24 h；C：48 h；D：72 h
图 6 转染后 RPMI8226 细胞的 survivin、MMP-2、MMP-9、ARK5、cyclin B1蛋白表达的差异
Figure 6. The expression of survivin, MMP-2, MMP-9, ARK5, and cyclin B1 protein in RPMI8226 cells after transfection
图 7 转染后RPMI8226细胞的Caspase-3/7蛋白活性的差异
Figure 7. The change of Caspase-3/7 protein activity in RPMI8226 cells after transfection
*P<0.05 c-maf siRNA group compared with other two groups
表 1 c-maf siRNA抑制 c-maf 基因的表达
Table 1. siRNA inhibits c-maf mRNA expression in RPMI8226 cells
表 2 转染后不同时间G2/M期细胞数量比例 （%）
Table 2. The percentage of RPMI8226 cells in G2/M phase at different time points（%）
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