Li Juan, Zhao Ying, Luo Shao-Kai, Zhang Dian-Bao, Huang Bei-Hui
Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510080, P. R. China.
Ai Zheng. 2006 Dec;25(12):1488-92.
BACKGROUND & OBJECTIVE: Survivin, a member of the inhibitors of apoptosis protein family (IAPs), is overexpressed in many cancers, but not in normally differentiated adult tissues. It is known to be involved in poor prognosis and resistance to chemotherapy and radiotherapy in many cancers. This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human myeloma cell line KM3, observe its effects on apoptosis of KM3 cells and sensitivity of KM3 cells to adriamycin (ADM).
Small interfering RNA (siRNA) targeting survivin mRNA was designed and in vitro chemosynthesized, and transfected into KM3 cells. Survivin mRNA and protein levels in KM3 cells were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot 24, 48, and 72 h after transfection. The apoptosis of KM3 cells was observed under fluorescent microscope before and after transfection. The sensitivity of KM3 cells to ADM before and after transfection was assessed.
The mRNA level of survivin was down-regulated by (47.0+/-4.3)%, (81.4+/-6.2)%, and (49.1+/-7.9)% of control at 24, 48, and 72 h after siRNA transfection, respectively; while the protein level of survivin was down-regulated by (39.6+/-3.8)%, (56.4+/-6.7)%, and (68.2+/-5.1)% of control, respectively. Under fluorescent microscope, the apoptosis rate of KM3 cells was (28+/-7)% at 48 h after transfection of survivin siRNA, which was significantly higher than that of control cells after transfection of negative siRNA (P<0.05). The 50% inhibition concentration (IC(50)) of ADM to KM3 cells was decreased from (1.12+/-0.14) micromol/L to (0.31+/-0.03) micromol/L.
Down-regulation of survivin expression in KM3 cells by siRNA could effectively induce apoptosis of KM3 cells and increase their sensitivity to ADM.
生存素是凋亡抑制蛋白家族（IAPs）的成员之一，在多种癌症中过度表达，但在正常分化的成人组织中不表达。已知其与多种癌症的预后不良及化疗和放疗耐药有关。本研究旨在利用RNA干扰技术下调人骨髓瘤细胞系KM3中生存素基因的表达，观察其对KM3细胞凋亡及KM3细胞对阿霉素（ADM）敏感性的影响。
设计并体外化学合成靶向生存素mRNA的小干扰RNA（siRNA），转染入KM3细胞。在转染后24、48和72小时，使用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测KM3细胞中生存素mRNA和蛋白质水平。在转染前后，在荧光显微镜下观察KM3细胞的凋亡情况。评估转染前后KM3细胞对ADM的敏感性。
siRNA转染后24、48和72小时，生存素的mRNA水平分别下调至对照的（47.0±4.3）%、（81.4±6.2）%和（49.1±7.9）%；而生存素的蛋白质水平分别下调至对照的（39.6±3.8）%、（56.4±6.7）%和（68.2±5.1）%。在荧光显微镜下，转染生存素siRNA后48小时，KM3细胞的凋亡率为（28±7）%，显著高于转染阴性siRNA后的对照细胞（P<0.05）。ADM对KM3细胞的50%抑制浓度（IC50）从（1.12±0.14）μmol/L降至（0.31±0.03）μmol/L。
通过siRNA下调KM3细胞中生存素的表达可有效诱导KM3细胞凋亡并增加其对ADM的敏感性。
