沉默survivin基因以增加骨髓瘤细胞对阿霉素敏感性的研究

Effect of Silencing Survivin Gene on Sensitivity of Myeloma Cells to Adriamycin

背景与目的:Survivin是凋亡抑制蛋白家族成员之一,在多种恶性肿瘤中高表达,但在正常成人分化组织中无表达。其表达与多种肿瘤的预后及肿瘤对放、化疗的耐受有关。本研究采用RNA干扰技术下调survivin基因在人骨髓瘤细胞系KM3中的表达,观察其诱导KM3细胞凋亡的作用,及是否增加KM3细胞对阿霉素的敏感性。方法:针对survivinmRNA体外化学合成小干扰RNA(siRNA),采用脂质体介导转染KM3细胞,以RT-PCR和Westernblot方法检测转染后24h、48h和72hKM3细胞survivinmRNA和蛋白的表达,荧光显微镜观察干扰前后细胞的凋亡情况,细胞毒分析方法比较转染前后对阿霉素敏感性的变化。结果:转染survivinsiRNA后24h、48h和72h,与阴性对照组相比,KM3细胞survivinmRNA水平分别下调了(47.0±4.3)%、(81.4±6.2)%和(49.1±7.9)%,蛋白质表达下调了(39.6±3.8)%、(56.4±6.7)%和(68.2±5.1)%。荧光显微镜下可见转染survivinsiRNA48h后的KM3细胞,凋亡率为(28±7)%,明显高于转染阴性对照siRNA的细胞。转染后细胞对阿霉素的敏感性增加,IC50由(1.12±0.14)μmol/L下降至(0.31±0.03)μmol/L。结论:以siRNA下调survivin的表达可有效诱导KM3细胞凋亡,并增加其对阿霉素的敏感性。

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）. METHODS： 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. RESULTS： 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 （IC5o） of ADM to KM3 cells was decreased from （1.12±0.14） μmol/L to （0.31±0.03） μmol/L. CONCLUSION： Down-regulation of survivin expression in KM3 cells by siRNA could effectively induce apoptosis of KM3 cells and increase their sensitivity to ADM.