PLK1基因siRNA的构建及对甲状腺未分化癌细胞增殖和凋亡的影响

Construction of PLK1 siRNA and its effects on proliferation and apoptosis of undifferentiated human thyroid cancer cells

目的构建保罗样激酶-1（PLK1）基因小干扰RNA（siRNA），并观察其对甲状腺癌细胞增殖和凋亡的影响。方法设计合成5个PLK1siRNA（S1、S2、S3、S4、S5），转染人甲状腺癌ARO细胞后，采用实时定量RT—PCR方法筛选下凋PLK1mRNA效果最好的siRNA；然后以该siRNA转染ARO细胞后，分别以实时定量RT—PCR和Western blot检测各组细胞PLK1基因表达情况；以MTT法检测对各组细胞增殖的影响；分别以caspase-3活性和TUNEL方法明确甲状腺癌细胞凋亡情况。结果5个siRNA均明显下调PLK1mRNA水平，以S4效果最好，抑制率达91．5％。MTT结果显示，S4siRNA转染对甲状腺癌细胞增殖均有明显的抑制作用，且呈浓度和时间依赖性（P〈0．05）。与对照组比较，S4siRNA转染组癌细胞caspase-3活性明显升高，且呈浓度和时间依赖性（r=0．919；r=0．957）；TUNEL结果显示，S4 siRNA转染组出现明显的凋亡现象，且呈浓度依赖性（r=0．932）。结论PLK1基因在甲状腺未分化癌细胞增殖中具有重要的调控作用。PLK1 siRNA转染可明显抑制癌细胞增殖，诱导凋亡是其重要机制之一。

Objective To study effects of polo-like kinase-1 （PLK1） small interfering RNA （siRNA） on proliferation and apoptosis of undifferentiated human thyroid cancer cells. Methods 5 PLK1 siRNA （ S1, S2, S3, S4 and S5） were constructed and used to transfect human thyroid cancer cell line ARO. RT-PCR was employed to pick out the most effective siRNA, which was then used to transfect ARO cell. RT-PCR and western blot were used to detect PLK1 expression in thyroid cancer cells, which were divided into different groups. MTT assay was performed to examine the effects of PLK1 siRNA on thyroid cancer cells in all groups. Apoptosis of thyroid cancer cells was observed by caspase-3 activity and TUNEL. Results All the 5 siRNA down-regulated PLK1 mRNA expression, among which S4 showed the best effect. S4 transfection could obviously inhibit proliferation of thyroid cancer cells in dose and time dependent manner. Compared with control groups, caspase-3 activity of cancer cells in S4 transfected group increased significantly. The effect of S4 transfection was dose and time dependent. TUNEL results showed apoptosis of cancer cells transfected by S4 siRNA was obvious and apoptosis of cells was dose-dependent. Conclusions PLK1 may play an important role in proliferation of undifferentiated thyroid carcinoma. PLK1 siRNA transfection can inhibit proliferation of throid cancer cell through apoptosis induction.