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靶向c-myc基因小RNA干扰对胆囊癌细胞侵袭运动的影响
The siRNA Targeted to c-myc Affect Gallbladder Cancer Cells’ Migration and Invasion Ability
【作者】 蔡民；
【导师】 李立波；
【作者基本信息】 浙江大学 ， 外科学， 2010， 硕士
【摘要】 目的：通过观察c-myc对胆囊癌细胞运动和侵袭能力的影响,以论证c-myc在肿瘤侵袭和转移中的作用,为胆囊癌侵袭和转移干预提供分子靶点。方法：采用实时定量PCR和western blot检测亲代和高转移胆囊癌细胞系中c-myc基因表达,设计靶向c-myc的小RNA[small interference RNA,siRNA]转染高转移的胆囊癌细胞,通过体外侵袭实验研究c-myc siRNA对胆囊癌高转移细胞迁移和侵袭能力的影响。此外,通过western blot方法检测转染c-myc siRNA后c-myc下游部分信号通路的变化,进一步研究c-myc siRNA抑制胆囊癌细胞转移的机制。结果：通过实时定量PCR检测发现,c-myc的RNA水平在GBC-SD/M3细胞中是其在GBC-SD细胞中的2.18倍(p=0.0019)；在蛋白水平上,c-myc在GBC-SD/M3中的表达也高于其在GBC-SD中的表达。靶向c-myc的siRNA转染GBC-SD/M3细胞后,与对照相比,c-myc siRNA能够有效地抑制c-myc蛋白在GBC-SD/M3细胞中的表达。与对照相比,c-myc siRNA可以有效的抑制GBC-SD/M3细胞的迁移(p=0.0168)。对GBC-SD/M3细胞的侵袭能力检测后发现,c-myc siRNA同样也能够有效的抑制GBC-SD/M3细胞的侵袭(p=0.0005)。进一步检测c-myc siRNA对LIN28蛋白水平的影响,表明c-myc siRNA转染后,LIN28的蛋白水平也相应下调,let-7a的表达水平有所上升,从而抑制肿瘤细胞的转移。结论：c-myc在高转移的胆囊癌细胞中表达上调。通过siRNA下调其表达后可以抑制胆囊癌细胞的转移和侵袭,这种抑制作用可能与LIN28的下调有关。
【Abstract】 objective The role of c-myc gene in metastasis of gallbladder cancer was investigated by observation of the mobility and invasion of gallbladder cancer cells in vitro, in order to provide the evidences for selecting molecular targets in interfering gallbladder cancer metastasis.Methods the expression of c-myc gene was determined by real time PCR and western blot in parental gallbladder cancer cell line GBC-SD and its highly metastatic subpopulation GBC-SD/M3. The siRNA targeted to c-myc were transfected into GBC-SD/M3, and then the migration and invasion abilities of GBC-SD/M3 cells were detected by in vitro transwell assays. The changes of the downstream signal of c-myc were determined by western blot assays.Results the expression of c-myc gene was up-regulated in highly metastatic subpopulation, compared with that in its parental gallbladder cancer cell line. After the transfection of the c-myc siRNA in highly metastatic subpopulation, the RNA and protein levels were significantly reduced. Meanwhile, the migration and invasion abilities of the GBC-SD/M3 cells were inhibited. The detection of the downstream signal pathway of c-myc indicated LIN28 was reduced after the transfection of c-myc siRNA.Conclusion c-myc was up-regulated in the GBC-SD/M3 compared with GBC-SD. c-myc siRNA reduced the migration and invasion of gallbladder cancer cells. The inhibition may involve the down-regulation of LIN28.