MSI-2小干扰RNA对急性髓系白血病细胞株THP-1细胞增殖及NUMB表达的影响

Influence of RNA interference on MSI-2 gene in THP-1 cell and expression of NUMB

目的：探讨Musashi-2（MSI-2）基因小干扰RNA（siRNA）对急性髓系白血病细胞株THP-1细胞增殖的抑制作用及其下游通路基因NUMB表达的影响。方法选择针对MSI-2基因三种不同的siRNA片段，在脂质体介导下转染THP-1细胞，采用MTT法、集落形成实验及AnnexinⅤ/PI法分别观察沉默MSI-2基因后对细胞增殖、集落生成和细胞凋亡的影响；Western blot法检测caspase-3、PARP和NUMB蛋白表达水平。结果 HSS 133730片段干扰后MSI-2 mRNA及蛋白水平的降低最明显。转染MSI-2 siRNA 24 h后，THP-1细胞增殖抑制率为（47.89±7.64）%，明显高于阴性对照组（无关序列）（P=0.005）。培养9 d后，MSI-2 siRNA组细胞集落数为7.50±1.53，明显低于阴性对照组的35.75±7.46（P＆lt;0.001）；转染MSI-2 siRNA 24和48 h后，THP-1细胞凋亡率分别为（15.22±1.52）%和（33.83±3.96）%，均明显高于阴性对照组的（9.67±1.27）%和（12.42±2.07）%（P值分别为0.008和0.001），并伴随caspase-3和PARP剪切增加；同时也观察到潜在下游基因NUMB上调。结论沉默MSI-2基因能抑制THP-1细胞增殖，诱导细胞凋亡；上调NUMB可能是其分子作用机制之一。

Objective To investigate the effect of small interfering RNA（siRNA）for MSI-2 on the growth, apoptosis and NUMB expression of THP-1 cells. Methods Three siRNA for MSI-2 gene was designed and transfected into THP-1 cells. The cell inhibition, colony formation and apoptosis were determined. The protein expression of NUMB, caspase-3 and PARP were detected by Western blotting. Results After MSI-2 expression of THP-1 cells was down-regulated for 24 hours, cell inhibition of siRNA MSI-2 group was（47.89±7.64）%, obviously higher than that of negative control group（P=0.005）. After 9 days, cell colony count of siRNA MSI-2 group was 7.50 ± 1.53, also lower than that of negative control group （35.75 ± 7.46, P〈0.001）. In addition, apoptotic rates of siRNA MSI-2 group at 24 hours [（15.22±1.52）%]and 48 hours[（33.83±3.96）%]were significantly higher than those of negative control group（P=0.008 and P=0.001, respectively）. Accordingly, activations of caspase-3 and PARP and increased NUMB were observed in siRNA MSI-2 group. Conclusion siRNA for MSI-2 gene could increase the expressions of NUMB to inhibit the proliferation and induce apoptosis of THP-1 cells.