Song Hui, Xin Xiao-Yan, Xiao Feng, Wang De-Tang, Yue Qiao-Hong, Han Xing
Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, Shannxi, Xi'an 710032, China.
Eur J Obstet Gynecol Reprod Biol. 2008 Jan;136(1):83-9. doi: 10.1016/j.ejogrb.2006.07.057. Epub 2006 Nov 13.
Survivin is a new member of the inhibitors of apoptosis (IAPs) family. It is upregulated in various malignancies including human cervical carcinomas. Reduction of this molecule has resulted in chemosensitization, but it is uncertain whether it can lead to radiosensitization. We observed the effect of survivin gene RNA interference (RNAi) on the proliferation, apoptosis, and radiosensitivity of the human cervical carcinoma cells HeLa.
Human cervical carcinoma cells (HeLa) were transfected with the specific siRNA expression vector (pSilencer2.1-s2) designed to target survivin mRNA. A corresponding site-mutated vector was constructed as a negative control (pSilencer2.1-NC). The expression of survivin mRNA and its protein among the stable transfected cells and the untransfected ones was detected by semi-quantitative RT-PCR and Western blotting respectively. The cell growth was examined by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle distribution and cell apoptosis were measured by flow cytometry. The changes in cell radiosensitivity were observed by clonogenic survival assay.
Three stable transfected cell lines: HeLa-s2 (with pSilencer2.1-s2), HeLa-NC (with pSilencer2.1-NC), and HeLa-U6 neo (with empty vector pSilencer2.1-U6 neo) were established. The expression levels of survivin gene mRNA and protein in HeLa-s2 were significantly lower than in HeLa-NC, HeLa-U6 neo, and those untransfected HeLa cells. The expression inhibitory rates were 62.8% and 60.1%. The cell proliferation of HeLa-s2 was inhibited, and the highest inhibitory rate was 57.8+/-2.1%. The changes in cell cycle distribution in HeLa-s2 compared with the other three cell lines were obvious, many cells were blocked in the G(0)/G(1) phase 72.7+/-3.1% (P<0.05), reduced sharply in the G(2)/M phase (5.1+/-2.9)% (P<0.05), and also the apoptotic rate was 29.2+/-1.4%, obviously increasing (P<0.05). At the same dose of radiation, the cloning efficiency of HeLa-s2 declined notably (P<0.05); the cell survival curve showed a significant decrease in D(0) and D(q), which were 3.15 and 1.21, respectively (P<0.05), and the radiation enhancement ratios were 2.01 (a ratio of D(0)) and 1.77 (a ratio of D(q)).
Survivin gene RNAi not only could inhibit the proliferation of human cervical carcinoma cells (HeLa), but also could significantly enhance the radiosensitivity of those cells through the reduction of its mRNA and protein. Therefore, an RNAi-targeted survivin gene strategy would be a potential approach to radiosensitization therapy in human cervical carcinomas.
生存素是凋亡抑制蛋白（IAPs）家族的新成员。它在包括人类宫颈癌在内的多种恶性肿瘤中上调。该分子的减少已导致化学增敏作用，但它是否能导致放射增敏尚不确定。我们观察了生存素基因RNA干扰（RNAi）对人宫颈癌细胞HeLa增殖、凋亡及放射敏感性的影响。
用设计用于靶向生存素mRNA的特异性小干扰RNA（siRNA）表达载体（pSilencer2.1-s2）转染人宫颈癌细胞（HeLa）。构建相应的位点突变载体作为阴性对照（pSilencer2.1-NC）。分别通过半定量逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测稳定转染细胞和未转染细胞中生存素mRNA及其蛋白的表达。通过噻唑蓝（MTT）法检测细胞生长情况。通过流式细胞术测量细胞周期分布和细胞凋亡情况。通过克隆形成存活试验观察细胞放射敏感性的变化。
建立了三个稳定转染细胞系：HeLa-s2（含pSilencer2.1-s2）、HeLa-NC（含pSilencer2.1-NC）和HeLa-U6 neo（含空载体pSilencer2.1-U6 neo）。HeLa-s2中生存素基因mRNA和蛋白的表达水平显著低于HeLa-NC、HeLa-U6 neo及未转染的HeLa细胞。表达抑制率分别为62.8%和60.1%。HeLa-s2的细胞增殖受到抑制，最高抑制率为57.8±2.1%。与其他三个细胞系相比，HeLa-s2的细胞周期分布变化明显，许多细胞阻滞于G(0)/G(1)期，比例为72.7±3.1%（P<0.05），G(2)/M期比例急剧下降（5.1±2.9）%（P<0.05），凋亡率为29.2±1.4%，明显升高（P<0.05）。在相同剂量辐射下，HeLa-s2的克隆效率显著下降（P<0.05）；细胞存活曲线显示D(0)和D(q)显著降低，分别为3.15和1.21（P<0.05），放射增强比分别为2.01（D(0)比值）和1.77（D(q)比值）。
生存素基因RNAi不仅能抑制人宫颈癌细胞（HeLa）的增殖，还能通过降低其mRNA和蛋白水平显著增强这些细胞的放射敏感性。因此，以RNAi靶向生存素基因的策略可能是人类宫颈癌放射增敏治疗的一种潜在方法。
