RNAi沉默Plk1对胰腺癌细胞系AsPC-1侵袭转移的影响

Influence of Plk1 blocking with RNAi in invasiveness and metastasis in pancreatic carcinoma cell line of AsPC-1

目的探讨RNAi沉默Plk1基因表达与胰腺癌细胞侵袭转移能力的关系。方法根据Plk1基因特点,设计并用化学方法合成了4个小干扰核糖核酸分子(siRNA)(shplk1-1、shplk1-2、shplk1-3、shplk1-4),脂质体转染法将短链siRNA转入AsPC-1细胞;细胞分为:未处理组为对照组、空质粒组、shplk1-1组、shplk1-2组、shplk1-3组、shplk1-4组。采用RT-PCR和Western blot法检测siRNA对Plk1表达的抑制效果;采用Transwell法检测转染后细胞侵袭能力;采用划痕实验检测转然后细胞迁移能力。结果 RT-PCR和Western blot结果示所设计的4个siRNA均能明显抑制AsPC-1细胞Plk1 mRNA水平,以shplk1-3效果最好,与对照组、空质粒组及其他三组比较,转染shplk1-3组Plk1 mRNA和蛋白的表达水平显著降低(P<0.01);以转染shplk1-3处理AsPC-1细胞后与对照组、空质粒组比较,Transwell侵袭实验证实:细胞转染24 h后,正常对照组、空质粒组和shplk1-3组穿过人工基底膜的侵袭细胞数分别为(195±16)、(176±13)、(83±5)个,侵袭能力明显降低,差异有高度统计学意义(P<0.01);细胞划痕实验证实:shplk1-3组迁移能力明显降低。结论抑制胰腺癌细胞Plk1基因,侵袭迁移能力明显降低,能够为胰腺癌的治疗提供新的策略。

Objective To investigate the regulatory role of polo-like kinase-1 （Plk1） gene in the invasion and migration of pancreatic cancer cells.Methods 4 small interfering RNAs （siRNAs） （shplk1-1,shplk1-2,shplk1-3,shplk1-4） targeting Plk1 gene were designed and synthesized according to Plk1 mRNA sequence.The cells were divided into control group,empty plasmid group,shplk1-1 group,shplk1-2 group,shplk1-3 group and shplk1-4 group.Mter transfection of AsPC-1 cells with the siRNAs,real-time RT-PCR and Western blotting were performed to examine the changes in Plk1 gene expression in the cells.Transwell assay was used to explore the invasion ability of the cells.Cell migration was assessed by a wound healing assay.Results RT-PCR and Western blot results showed that all the 4 siRNAs were capable of suppressing Plk1 mRNA expression in AsPC-1 cells,among which the AsPC-1 siRNA had the strongest effect,the differences were statistically significant （P 〈 0.01）.The transwell and the wound healing assays showed that the migratory and invasive capacity of AsPC-1 cells transfected with AsPC-1 reduced to （195±16）,（176±13）,（83±5）,the differences were statistically significant （P 〈 0.01）.Transfer ability of shplk1-3 group was reduced according to the wound healing assay.Conclusion Blocking Plk1 activation with shplk1 can inhibit the invasion and metastasis ability of pancreatic cancer cells in vitro through down-regulation of plk1 expression.Blocking Plk1 may provide a novel strategy in prevention of invasion and metastasis of pancreatic cancer.