靶向survivin的siRNA对肝癌HepG2细胞增殖和转移能力的影响

Survivin-targeted siRNA inhibits proliferation of liver cancer cell lineHepG2 in vitro and in vivo

目的探讨针对人survivin基因的siRNA对肝癌细胞HepG2的体内外抑制作用。方法合成survivin基因的RNA干涉(RNA interferance,RNAi)特异性片段,构建survivin特异性的RNA干涉载体pSiS,转染HepG2细胞,G418筛选稳定转染的细胞系。细胞计数检测细胞生长情况;Western blot-ting检测细胞中survivin蛋白表达的变化;通过AnnexinV法染色和流式细胞术检测细胞凋亡;观察裸鼠皮下HepG2移植瘤的形成。结果成功构建了survivin基因siRNA真核表达载体pSiS,获得了稳定转染的细胞HepG2/pSiS。与HepG2、HepG2/pSi(空质粒对照细胞)相比,HepG2/pSiS细胞生长曲线十分平缓,差异有统计学意义(P<0.01);Western blotting显示,HepG2/pSiS细胞中survivin蛋白表达减少,细胞凋亡率增加(P<0.01)。HepG2/pSiS移植瘤形成明显受到抑制。结论Survivin特异性siRNA可明显促进肝癌细胞HepG2的凋亡,抑制该肿瘤细胞体外生长和体内移植成瘤。

Objective To explore the inhibitory effect of survivin-targeted siRNA on liver cancer cell line HepG2 in vivo and in vitro. Methods According to the survivin cDNA sequence, the specific RNA interference （RNAi） fragmentswere designed and synthesized, which were then cloned into pSileneer 3. 02H1 neo plasmid vector to construct pSiS vector. HepG2 cellswere transfected with RNAi vectors and negative control vector separately; the stably transfected cell strains were selected by G418; and cell growth was assessed by cell counting. Expression of survivin protein was investigated byWestern blotting. Apoptosis analysiswas examined byAnnexin V method. Tumorigenesis assaywas conducted in nudemice. Results The specific survivin-targeted siRNA eukaryotic vectorpSiSwas successfully constructed, and transfectants were obtained. Compared with blank vector transfected cells and untransfected cells, HepG2/pSiS cells had a slow growth （ P 〈 0. 01 ）. Expression of survivin protein was significantly inhibited inHepG2/pSiS cells, which had an increased apoptotic rate （ P 〈 0. 01 ）. The tumorigenesis of transplanted HepG2/pSiS was greatly inhibited. Conclusion Survivintargeted siRNA can promote the apoptosis of HepG2 cells, and inhibit the in vitro growth and in vivo tumor formation in nude mice.