siRNA沉默KAP-1表达抑制胰腺癌细胞PANC-1的侵袭能力  

SiRNA-mediated silencing of KAP-1 expression inhibits invasion of human pancreatic cancer PANC-1 cells

作　　者：江建新[1] 詹磊[1] 黄洋[1] 何燕浙[1] 孙诚谊[1] 

机构地区：[1]贵阳医学院附属医院肝胆外科,贵州省贵阳市550001

出　　处：《世界华人消化杂志》2013年第19期1859-1865,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.81160311;贵州省科教青年英才培养工程基金资助项目;No.黔省专合字[2012]177号;贵州省科技厅2011年度社会攻关计划基金资助项目;No.黔科合SY字[2011]3007;贵州省肝胰疾病研究科技创新人才团队基金资助项目;No.黔科合人才团队[2010]4010~~

摘　　要：目的:观察siRNA沉默KAP-1基因表达对人胰腺癌PANC-1细胞侵袭能力的影响,探求该基因作为治疗靶点的可行性.方法:针对KAP-1基因设计5条siRNA,构建真核表达载体,转染293T细胞筛选RNAi有效靶点,包装成重组慢病毒Lv-siRNA-KAP-1,感染胰腺癌细胞PANC-1成功后RT-qPCR检测RNA干扰沉默效果,Transwell小室检测细胞侵袭能力.Western blot检测波形蛋白表达.结果:转染48h后,5条siRNA能显著抑制KAP-1的蛋白表达;其中最有效的1条siRNA包装成重组慢病毒Lv-siRNA-KAP-1,感染PANC-1细胞侵袭能力明显受到抑制;感染后细胞侵袭数目(97.3±25.6)较空白对照组(253.3±20.6)与阴性对照组(213.2±19.4)明显减少,差异显著(P<0.05);感染后PANC-1细胞的波形蛋白表达下调.结论:Lv-siRNA-KAP-1能显著抑制PANC-1细胞KAP-1的表达,抑制PANC-1细胞侵袭能力及波形蛋白表达,KAP-1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 KAP-1 gene silenc- ing on invasion of human pancreatic cancer PANC-1 cells, and to explore the feasibility of human KAP-1 gene as a therapeutic target for pancreatic cancer. METHODS： Five KAP-l-specific siRNAs weredesigned using online software to construct pGC-LV-siRNA-KAP-1 plasrnid. Western blot was used to screen the efficient plasmid in 293T cells, and the most efficient one was packed into the recombinant lentivirus Lv-siRNA-KAP-1 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing ef- fect of Lv-siRNA-KAP-1 in PANC-1 cells was validated by real-time PCR. After PANC-1 cells were infected with Lv-siRNA-KAP-1, cell inva- sion was detected by Transwell chamber assay. Vimentin expression in cells was detected by Western blot. RESULTS： Four KAP-l-specific siRNAs could silence the expression of KAP-1 at the protein level 48 h after transfection. The most efficient pGC-siRNA-3 plasmid was used to construct Lv-VIM-shRNA. The titer of lentivirus was 2×109 TU/mL. KAP-1 knockdown significantly inhibited invasion of human pancreatic cancer PANC-1 cells compared to blank control and negative control cells （97.3±25.6 vs 253.3± 20.6, 213.2± 19.4, both P 〈 0.05）. The expression of vimentin was down-regulated in PANC-1 cells infected with Lv-siRNA-KAP-1. CONCLUSION： Lv-siRNA-KAP-1 could effec- tively inhibit the expression of KAP-1 gene in PANC-1 cells in vitro, cell invasion and Vimentin expression. KAP-1 might serve as a new target for gene therapy of pancreatic cancer.

关 键 词：胰腺癌 KAP-1 小干扰RNA 侵袭 

分 类 号：R735.9[医药卫生—肿瘤]