siRNA沉默RPL31表达对胰腺癌PANC-1细胞增殖的抑制作用  

SiRNA-mediated silencing of the RPL31 gene inhibits proliferation of human pancreatic cancer PANC-1 cells

作　　者：李朝东[1] 戈梅[2,3] 罗敏玉[3] 陈代杰[1] 

机构地区：[1]华东理工大学生物工程学院,上海市200237 [2]上海交通大学药学院,上海市200240 [3]上海来益生物药物研究开发中心有限责任公司,上海市201203

出　　处：《世界华人消化杂志》2012年第30期2895-2901,共7页World Chinese Journal of Digestology

基　　金：重大新药创制-微生物药物技术创新与新药创制产学研联盟基金资助项目;No.2010ZX09401-403~~

摘　　要：目的:观察siRNA沉默核糖体蛋白L31(ribosomal protein L31,RPL31)基因表达对人胰腺癌PANC-1细胞的影响.方法:针对RPL31基因设计了3条siRNA,使用脂质体LipofectamineTM2000转染人胰腺癌PANC-1细胞,同时设立空白对照组和阴性对照组,转染成功后通过实时定量PCR及Western blot分别从mRNA和蛋白水平检测RNA干扰沉默RPL31基因的效果,四氮唑盐比色法(MTT)检测细胞增殖,流式细胞术检测细胞周期分布,Transwell小室检测细胞迁移,ELISA检测血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达.结果:转染48h后,3条siRNA能显著抑制RPL31基因mRNA和蛋白的表达;MTT结果显示,PANC-1细胞增殖明显受到抑制;细胞周期分布中G0/G1期明显增加、S期减少(G0/G1:59.85%±5.47%vs45.71%±3.44%;S:28.63%±4.52%vs45.13%±2.64%,均P<0.05);转染后细胞迁移数目(178.6个±30.3个)较空白对照组(470.5个±22.8个)与阴性对照组(474.2个±20.4个)明显减少,差异显著(P<0.05);转染后PANC-1细胞的VEGF表达量也明显减少[1563.45pg/(106cell24h)±24.95pg/(106cell24h)vs2804.6pg/(106cell24h)±40.46pg/(106cell24h),2791.5pg/(106cell24h)±44.77pg/(106cell24h)].结论:RPL31siRNA能明显抑制PANC-1细胞RPL31的表达,抑制细胞增殖,降低细胞迁移及VEGF表达,RPL31基因有可能成为胰腺癌基因治疗的新靶点.AIM： To investigate the impact of small interfer- ing RNA （siRNA）-mediated RPL31 gene silenc- ing on biological behavior of human pancreatic cancer PANC-1 cells, and to explore the feasibil- ity of using the human RPL31 gene as a thera- peutic target for pancreatic cancer.METHODS： Three RPL31-specific siRNAs were designed and transfected into PANC-1 cells us- ing LipofectamineTM 2000. Blank control andnegative control groups were run at the same time. After PANC-1 cells were transfected with RPL31-specific siRNA, the levels of RPL31 mRNA and protein were detected by quantitative real- time PCR （qRT-PCR） and Western blot, respec- tively. Cell proliferation was detected by MTT assay. Cell cycle progression was determined by flow cytometry. Cell migration was determined by Transwell chamber assay. Vascular endothe- lial growth factor （VEGF） expression in cells was detected by ELISA.RESULTS： All three RPL31-specific siRNAs could silence the expression of RPL31 at the mRNA and protein levels 48 hours after transfection. MTT as- say showed that cell proliferation was significant- ly inhibited. Flow cytometry analysis revealed that PANC-1 cells transfected with RPL31 siRNA had a more significant cell cycle arrest （G0/G1 phase： 59.85% ± 5.47% vs 45.71% ± 3.44%; S phase： 28.63% ± 4.52% vs 45.13% ± 2.64%, both P 〈 0.05）. RPL31 knockdown significantly suppressed VEGF expression （1563.45 ± 24.95 pg/106 cells/24 h vs 2804.6± 40.46 pg/106 cells/24 h, 2791.5± 44.77 pg/106 cells/24 h, both P 〈 0.05） and the migra- tion of PANC-1 cells （178.6± 30.3 vs 470.5± 22.8, 474.2 ± 20.4, both P 〈 0.05） compared to the blank control and negative control groups.CONCLUSION： Transfection of RPL31-specific siRNAs could effectively inhibit RPL31 expression, significantly suppress cell proliferation, and reduce cell migration and VEGF expression. RPL31 might serve as a target for gene therapy of pancreatic cancer.

关 键 词：胰腺癌 核糖体蛋白L31 小干扰RNA 细 胞增殖 细胞迁移 

分 类 号：R735.9[医药卫生—肿瘤]