摘要
目的探讨RNA干扰下调survivin基因表达后腺样囊性癌细胞生物学行为变化及其与caspase-3基因表达的相关性。方法设计、合成小干扰RNA(small interfering RNA,siRNA),构建表达载体pGenesil-shRNA-survivin,将重组质粒导入人腺样囊性癌ACC-2细胞株,G418筛选阳性克隆。采用逆转录-聚合酶链反应(RT-PCR)、Western blot分别检测转染后的ACC-2细胞中survivin、caspase-3 mRNA及蛋白表达的变化,用噻唑蓝(MTT)法检测细胞的增殖,原位细胞凋亡(TUNEL)法及透射电子显微镜研究细胞凋亡。结果测序证实表达载体构建成功,在pGenesil-shRNA-survivin的ACC-2细胞株中,survivin mRNA及蛋白表达明显降低(P<0.05),caspase-3 mRNA及蛋白表达明显增加(P<0.05),survivin与caspase-3的mRNA和蛋白表达呈显著负相关(r=-0.333,P<0.05)。ACC-2细胞增殖受到抑制,透射电镜及TUNEL法可见细胞出现凋亡。结论成功构建了survivin基因的RNAi真核表达载体,pGenesil-shRNA-survivin重组质粒能有效抑制腺样囊性癌细胞生长。
Objective To investigate the effects of survivin down-regulation on the biological behavior of adenoid cystic carcinoma cells and the relationship with caspase-3. Methods Small interfering RNA （siRNA） was designed and synthesized for the construction of eukaryotic expression vector pGenesil-shRNA-survivin. The recombinant plasmid was transfected into ACC-2 cells. The positive clones were screened with G418. The expressions of survivin and caspase-3 mRNA and protein were detected by RT-PCR and Western blot. The proliferation of adenoid cystic carcinoma cells was detected by MTF. Cell apoptosis was detected by TUNEL assay and transmission electron microscopy. Results The recombinant plasmid was successfully constructed. Down-regulation was found in the expressions of survivin mRNA and protein （P 〈 0. 05 ） , but up-regulation in the expressions of caspase-3 mRNA and protein （P 〈 0. 05 ）. There was a negative correlation between the mRNA and protein expression of caspase-3 and survivin （ r = - 0. 333, P 〈 0. 05 ）. ACC-2 cell proliferation was inhibited. TUNEL assay and transmission electron microscopy showed typical apoptosis of cells. Conclusion The RNAi eukaryotic vector pGenesil-shRNA-survivin was constructed successfully. Recombinant plasmid pGenesil-shRNA-survivin can inhibit the proliferation of adenoid cystic carcinoma cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第11期1053-1056,共4页
Journal of Third Military Medical University
基金
重庆市自然科学基金(2007BB5064)~~
