沉默Bmi-1基因表达对胃癌细胞BGC823衰老和转移的作用  

ShRNA-mediated silencing of the Bmi-1 gene promotes senescence and inhibits migration in human gastric caner cell line BGC823

作　　者：高凤兰[1,2] 刘春灵[2] 李维山[2] 赵国强[1] 

机构地区：[1]郑州大学基础医学院,河南省郑州市450001 [2]漯河医学高等专科学校,河南省漯河市462002

出　　处：《世界华人消化杂志》2010年第4期335-339,共5页World Chinese Journal of Digestology

摘　　要：目的:研究靶向Bmi-1siRNA对胃癌BGC823细胞衰老和转移的作用.方法:设计Bmi-1的siRNA靶序列,分别合成两条互补的寡核苷酸链,退火后重组入pRNAT-U6.2载体,转化扩增后进行序列测定.用脂质体包裹转染人胃癌BGC823细胞,采用RT-PCR和Western blot分别检测Bmi-1基因mRNA和蛋白表达的变化.SA-β-Gal染色检测和细胞体外侵袭实验检测分析对细胞衰老和侵袭、转移的影响.结果:靶向Bmi-1基因的siRNA的双链寡核苷酸片段克隆入pRNAT-U6.2载体,经测序分析,插入片段正确;RT-PCR和Western blot检测显示,Bmi-1基因的表达水平明显降低,其中以1104-1122nt(GGAGGAGGTGAATGATAAA)为靶序列的siRNA沉默作用最佳,Bmi-1 mRNA和蛋白表达几乎完全抑制.转染siRNA组的衰老细胞百分率显著升高、穿透Matrigel的细胞数显著下降,与未转染和转染空载体组比较差异有显著性(P<0.01).结论:抑制胃癌BGC823细胞Bmi-1基因表达,可以增加细胞的衰老和降低细胞侵袭、转移的能力.AIM： To investigate the effects of B-cell-specific Moloney murine leukaemia virus insertion site 1 （Bmi-1） gene knock-down on cell senescence and migration in human gastric caner cell line BGC823. METHODS： Two pairs of complementary small hairpin RNA （shRNA） oligonucleotides targeting the Bmi-1 gene were devised, synthesized, annealed and cloned into the pRNAT-U6.2 vector. After DNA sequencing to verify the correct insertion of the shRNA sequences, the recombinant plasmids were transfected into BGC823 cells. The expression of Bmi-1 mRNA and protein was examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. The effects of Bmi-1 knockdown on cell senescence and migration were determined by β-Gal activity assay and Boyden chamber assay, respectively. RESULTS： The double-stranded shRNA oligonucleotides targeting the Bmi-1 gene were successfully cloned into the pRNAT-U6.2 vector. DNA sequencing results verified the correct insertion of the shRNA sequences. RT-PCR and Western blot analyses indicated that the expression levels of Bmi-1 mRNA and protein were significantly downregulated in cells transfected with the recombinant plasmids. Particularly, Bmi-1 protein expression was almost completely abolished in cells transfected with the recombinant vector harboring shRNA targeting the sequence GGAGGAGGTGAATGATAAA （nt 1 104-1 122）. Compared with untransfected cells and cells transfected with the empty vector, the average percentage of senescent cells increased and the number of cells passing through the Matrigel decreased in cells transfected with the recombinant vectors. CONCLUSION： ShRNA-rnediated silencing of the Bmi-1 gene can effectively promote cell senescence and reduce migration in human gastric caner cell line BGC823.

关 键 词：Bmi—1 SIRNA 胃癌 细胞衰老 细胞转移 

分 类 号：R735.2[医药卫生—肿瘤] R733.73[医药卫生—临床医学]