沉默BRCAA1基因对胃癌MGC-803细胞的抑制作用及其可能的机制  

Inhibitory effects of BRCAA1 gene silencing on gastric cancer MGC-803 cells and its possible mechanism

作　　者：刘彬[1] 崔大祥[1] 杜彤[1] 李智铭[1] 宋华[1] 杨浩[1] 鲍晨晨[1] 甘慧[1] 

机构地区：[1]上海交通大学微纳科学技术研究院生物纳米工程研究室微纳米制造技术国家重点实验室薄膜与微细技术教育部重点实验室,上海200240

出　　处：《中国肿瘤生物治疗杂志》2008年第6期522-526,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家重点基础研究发展(973)计划资助项目(No.2005CB723400-G);国家高技术研究发展(863)计划重点项目(No.2007AA022004);国家自然科学基金资助项目(No.30771075No.30672147);上海市科委基金资助项目(No.072112006-6)~~

摘　　要：目的:研究沉默乳腺癌相关抗原1(breast cancer-associated antigen 1,BRCAA1)基因对胃癌细胞株MGC-803的抑制作用及其可能的机制。方法:构建BRCAA1基因shRNA载体,将构建的shRNA-BRCAA1质粒与阴性对照质粒shRNA-N转染胃癌MGC-803细胞,24 h后用荧光显微镜观察转染效率,实时定量PCR检测BRCAA1和GAPDH基因mRNA表达水平MTT法检测转染后24、48与72 h的细胞增殖水平,Annxin-V PE/7AAD检测转染24 h后的细胞凋亡水平,Western blotting检测转染48 h后细胞的凋亡相关蛋白表达水平。结果:BRCAA1 siRNA表达质粒转染MGC-803细胞24 h的转染效率为(81.2±2.6)%。转染后48 h MGC-803细胞的BRCAA1 mRNA水平下降了61.4%,MGC-803细胞增殖的抑制率达45.0%,转染siRNA细胞的凋亡率明显高于未转染细胞和对照质粒转染细胞[(14.4±1.6)%vs(5.4±2.0)%,(4.4±2.5)5,P<0.05]。转染siRNA细胞的凋亡相关蛋白Rb与Bax的表达量显著增加(P<0.05),Bcl-2的表达量显著减少(P<0.05)。结论: BRCAA1基因的沉默可有效抑制人胃癌MGC-803细胞的增殖和诱导细胞凋亡,其机制与其促进Rb和Bax蛋白表达、抑制Bcl- 2蛋白表达有关。Objective ： To investigate the inhibitoR, effect of breast cancer-associated antigen 1 （ BRCAA1 ） gene silencing on gastric cancer MGC-803 ceils and the related mechanism. Methods： Plasmid shRNA-BRCAA1 and shRNA-N were constructed and transfected with FuGene HD into gastric cancer cell line MGC-803. The transfection efficiency was examined using fluorescent microscope 24 h later. The total RNAs was extracted 48 h after transfection and the expression of BRCAAI and GAPDH gene were analyzed by real-time PCR. The cell proliferation was assessed by MTT assay 24 h, 48 h, and 72 h after transfection. The cell apoptosis was determined by Annexin V-PE/7AAD. The expression of Rb, Bax, Bcl-2 and BRCAA1 proteins was analyzed by Western blotting 48 h after transfection. Results： We found that the transfection efficiency of shRNA-BRCAA1 was （81.2 ± 2.6） % 24 h after transfection. Forty-eight hours after transfection with shRNA-BRCAA1 the expression of BRCAA1 mRNA decreased by 61.4% ; the inhibition rate of MGC-803 cells growth was 45.0%. The cell apoptosis rate of shRNA-BRCAA1 transfection group was significantly higher than those of untransfected group and mock plasmid transfected group （ [ 14.4 ±1.6 ] % vs [ 5.4±2.0 ] %, [ 4.4 ± 2.5 ] % ,P 〈 0.05 ]. Cells transfected with shRNA-BRCAA1 had significantly increased expression of Rb and Bax proteins （ P 〈 0.05） , and decreased expression of Bcl-2 protein（P 〈0.05）. Conclusion： BRCAAI gene silencing can effectively inhibit the proliferation of MGC-803 cells and induce apoptosis, which might be related to the promotion of Rb and Bax proteins, and suppression of Bcl-2 protein.

关 键 词：乳腺癌相关抗原基因1 SHRNA 胃癌细胞 增殖 细胞凋亡 

分 类 号：R735.2[医药卫生—肿瘤] R730.54[医药卫生—临床医学]