特异性小干扰RNA沉默Polo样激酶1基因表达对恶性胶质瘤细胞增殖的影响

目的 探讨Polo样激酶1(PLK1)基因对胶质瘤细胞增殖的影响和可能机制. 方法 根据PLK1基因特点,设计并用化学方法合成了5个小干扰核糖核酸分子(siRNA)(P1、P2、P3、P4和P5).以这5个siRNA转染人胶质瘤TJ905细胞后.分别采用荧光实时定量RT-PCR和Western blot检测PLK1 mRNA和蛋白表达水平.分别采用MTT法和Western blot方法检测癌细胞增殖和增殖细胞核抗原(PCNA)蛋白水平,用TRA-.ELISA方法检测胶质瘤细胞端粒酶活性. 结果 所设计的5个siRNA均能明显抑制胶质瘤TJ905细胞PLK1 mRNA水平,以P4效果最好.以P4转染处理胶质瘤细胞后与脂质体对照组比较,PLK1基因mRNA水平和蛋白水平明显下调,差异有统计学意义(P均=0.000).MTT结果显示.与脂质体对照组比较P4 siRNA转染组癌细胞生长明显受到抑制,且呈浓度依赖性(r=0.868,P=0.000).Western blot结果显示,与脂质体对照组比较P4 siRNA转染组PCNA蛋白水平明显下降,差异有统计学意义(F=181.36,P=0.000).TRAP-ELISA结果显示,与脂质体对照组比较P4 siRNA转染组胶质瘤细胞端粒酶活性明显受到抑制,且呈浓度和时间依赖性(P=0.000). 结论 PLK1基因对胶质瘤细胞增殖具有重要的调控作用;以PLK1 siRNA转染处理胶质瘤细胞,可明显抑制胶质瘤细胞的恶性增殖,其机制可能与抑制端粒酶活性有关.

Effects of small interfering RNA targeting polo-like kinase-1 on the proliferation of human glioma cells in vitro

Objective To investigate the regulatory role of polo-like kinase-1 (PLK1) gene in the proliferation of human glioma cells. Methods Five small interfering RNAs (siRNAs) targeting PLK1 gene were designed and synthesized according to PLK1 mRNA sequence. After transfection of human glioma TJ905 cells with the siRNAs, real-time RT-PCR and Western blotting were performed to examine the changes in PLK1 gene expression in the cells. The growth of the transfected cells was evaluated by MTT assay and proliferating cell nuclear antigen (PCNA) protein expression determined using Western blotting. Telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA) was used to detect the changes in telomerase activity of the transfected cells. Results All the five siRNAs were capable of suppressing PLK1 mRNA expression in TJ905 cells, among which the P4 siRNA showed the strongest effect by reducing the PLK1 mRNA level by 93% 48 h after transfection at the concentration of 100 nmol/L. Compared with the oligofecamine control group cells the protein expression of PLK1 in TJ905 cancer cells transfected with P4 siRNA was also siguifieantly down-regulated. Transfection with P4 siRNA resulted in significant dose-dependent inhibitory effects on the proliferation and PCNA protein expression of TJ905 cells as compared to oligofecamine control group. The results of TRAP-ELISA showed obvious time- and dose-dependent inhibition of telomerase activity in the transfected cells as compared to oligofecamine control group. Conclusion PKL1 gene plays an important regulatory role in the proliferation of human glioma cells, and RNA interference of PLK1 gene can inhibit the cell proliferation possibly by suppressing the telomerase activity.