survivin基因RNA干涉对宫颈癌细胞凋亡和放射敏感性的影响

Influence of survivin gene-specific RNA interference on apoptosis and radiosensitivity of cervical carcinoma cells

目的：观察survivin基因siRNA干涉对宫颈癌细胞HeLa凋亡和放射敏感性的影响。方法：通过脂质体介导将含人survivin基因siRNA的重组真核表达质粒pSilencer2.1-s2转染宫颈癌细胞系HeLa,G418筛选阳性克隆,半定量PCR、Western blot分别检测survivin mRNA和蛋白表达,激酶活性检测法测定半胱氨蛋白水解酶-3(caspase-3)活性变化,流式细胞仪检测细胞凋亡情况,平板克隆形成实验、多靶单击模型拟合细胞存活曲线观察细胞放射敏感性变化。结果：G418筛选形成稳定转染阳性克隆,建立了3组稳定转染细胞系：HeLa-s2(转染重组质粒pSileneer2.1-s2)、HeLa-NC(转染阴性对照质粒pSi- lencer2.1-NC)和HeLa-U6 neo(转染空载质粒pSileneer2.1-U6 neo)。经与HeLa-NC、HeLa-U6 neo和未转染HeLa细胞比较,HeLa-s2 survivin mRNA和蛋白表达明显下降,与HeLa细胞相比,表达抑制率分别为：62.8%和60.1%；caspase-3激酶活性增强,D_(405)达1.26±0.04(P＜0.05)；细胞凋亡率为：(29.2±1.4)%,明显升高(P＜0.05)；同一剂量X线照射下,平板克隆形成率显著降低(P＜0.05)；细胞存活曲线显示：D_0、D_q值显著下降,分别为：3.15、1.21(P＜0.05),放射增敏比分别为：2.01 (D_0值比)、1.77(D_q值比)。结论：survivin基因siRNA干涉可通过阻抑HeLa细胞中survivin表达,增强caspase-3激酶活性,诱导细胞凋亡,并显著提高细胞的放射敏感性。

Objective：To observe the effect of survivin gene-specific RNA interference （RNAi） on apoptosis and radiosensitivity of cervical carcinoma HeLa cells. Methods： Human cervical carcinoma HeLa cells were transfected with the specific siRNA expression vector （pSilencer2. l-s2） designed to target survivin mRNA by using LipofectAMINE2000. The positive clones were selected with G418. The expressions of survivin mRNA and protein in the stable transfected cells and the untransfected ones were detected by semi-quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. The changes of caspase-3 activity were assessed by kinase activity test. Cell apoptosis was examined by flow cytometry. The changes of cell radiosensitivity were observed by clonogenic survival assay. Cell survival curves were calculated using the single-hit multi-target （SHMT） model. Results： Stable positive clones were obtained by screening with G418 for 34 d. Three stable transfected cell lines： HeLa-s2 （with recombined plasmid pSilencer2, l-s2）, HeLa-NC （with negative control pSilencer2.1-NC） and HeLa-U6-neo （with empty plasmid pSilencer2.1-U6-neo） were established. The expression levels of survivin mRNA and protein in HeLa-s2 cells were significantly lower than those in HeLa-NC, HeLa-U6-neo, and untransfected HeLa cells, with the expression inhibitory rate of 62.8% and 60.1%, respectively. The caspase-3 activity of HeLa-s2 cells increased notably and D40s was 1.26±0.04 （P〈0.05）. The apoptotic rate （29.2%±1.4% ） was elevated obviously （P〈0.05）. At the same dose of X ray irradiation, the cloning efficiency of HeLa-s2 cells declined sharply （P〈0.05）. The cell survival curve showed that Do （3.15） and Dq （1.21） was significantly decreased （P〈0.05） and the sensitive enhancement ratio （SER） was 2.01 for Do and 1.77 for Dq Conelusion：Survivin gene-specific siRNA interference enhanced radiosensitivity of HeLa cells by knocking down survivin gene expression, increasing caspase-3 activity, and inducing apoptosis.