RNA干扰技术抑制宫颈癌细胞低氧诱导因子-1表达的研究

HIF-1 Gene Expression Inhibited by RNAi in siHa Cervical Carcinoma Cells

目的:利用RNA干扰(RNAi)技术,使用特异的靶向作用HIF-1基因的小干扰RNA(siRNA)作用于宫颈癌细胞株SiHa,以了解siRNA对HIF-1基因的特异性抑制作用。方法:设计合成针对HIF-1的四个siRNA,鉴定无误后均借助脂质体转染宫颈癌细胞株siHa。检测转染前后常氧和低氧培养的SiHa细胞中mRNA和蛋白的表达变化,筛选出抑制率最高的siRNA。用筛选出的siRNA转染SiHa细胞,检测转染前后常氧和低氧培养的SiHa细胞中不同时间点mRNA和蛋白的表达变化,验证RNAi作用的特异性及时效性。结果:H2siRNA引起常氧和低氧培养的SiHa细胞中HIF-1基因表达水平的下降最显著(P<0.05);将H2siRNA转染常氧和低氧培养的SiHa细胞,转染后24、48、72及96h均可观察到HIF-1基因的显著降低,均以48h时靶基因表达水平的下降最显著(P<0.05),对应的非靶基因无明显抑制作用。结论:针对HIF-1构建的四个siRNA对HIF-1基因的干扰效率不同。本实验中,常氧及低氧培养SiHa细胞均以H2siRNA对HIF-1表达水平的降低作用最明显。将H2siRNA作用于SiHa细胞,可引起靶基因特异、高效性抑制,常氧及低氧条件下至少维持到转染后96h,且均以48h时基因表达水平的下降最显著。在SiHa细胞株,HIF-1siRNA通过下调HIF-1发挥抑制宫颈癌细胞生长的作用,RNAi技术可望为宫颈癌提供一种新的特异性基因治疗方法。

Objective： To utilize RNAi technology and observe the effect of siRNA specifically targeting the HIF-1 gene in SiHa human cervical carcinoma cells. Methods： We designed 4 separate siRNA＇s to target the HIF-1 gene and then detected the level of HIF-1 mRNA and protein under normoxic or hypoxic cultural conditions in order to screen for the most effective siRNA. The most effective siRNA was identified and transfected into SiHa cells that were cultured in normoxic or hypoxic conditions. We measured the expression level of HIF-1 mRNA and protein using RT-PCR and Western Blotting at 24h, 48h, 72h, and 96h after transfection. Results： According to our experiment, the most obvious decrease in target gene expression was caused by H2siRNA in both conditions. The HiF-1 expression level was reduced at 24h, 48h, 72h, and 96h after transfection in normoxic and hypoxic cultural conditions, especially at 48h. Conclusion： The 4 different siRNAs showed different inhibitory effects. The effect of siRNA is specific and efficient and can be sustained for at least 96h. HIF-1siRNA inhibited SiHa cell growth by downregulating HIF-1. RNAi may provide a new mode of gene therapy for human cervical carcinoma.