siRNA 抑制survivin 表达及其逆转人肺腺癌细胞耐药的机制。

Inhibition of survivin expression and mechanisms of reversing drug-resistance of human lung adenocarcinoma cells by siRNA.

机构信息

Department of Medical Oncology, Affiliated Tumor Hospital of Harbin Medical University, Harbin, Heilongjiang, China.

出版信息

Chin Med J (Engl). 2010 Oct;123(20):2901-7.

Abstract

BACKGROUND

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, overexpresses in tumor cells and not expresses in terminally differentiated adult tissues. This study aimed to investigate the effects of survivin-specific siRNA on cell proliferation, apoptosis and chemosensitivity to cisplatin in vitro and in vivo and explore the mechanisms about decreasing expression of survivin in reversing cancer cells resistance to chemotherapeutic drug.

METHODS

Survivin-specific siRNA was transfected into A549/DDP cells. The expression of survivin and lung resistance-related protein (LRP) mRNA levels were determined by RT-PCR, chemosensitivity of A549/DDP (cisplatin) cells to cisplatin was determined by MTT assay, and apoptosis and cell cycle were determined by flow cytometry (FCM). The protein expression levels of survivin, LRP, cyclin-D(1), caspase-3 and bcl-2 were determined by Western blotting analyses. The effect of survivin siRNA inhibition on tumor growth was studied in athymic nude mice in vivo.

RESULTS

Survivin-specific siRNA efficiently down-regulated survivin expression. The cell cycle was arrested at G2/M phase, and apoptosis was obviously found. Inhibition of survivin expression could make the IC50 and drug-resistant index of cisplatin decrease, and enhance the cancer cells sensitivity to cisplatin. After transfection by survivin-specific siRNA, expression of LRP and cyclin-D1 were downregulated, caspase-3 expression was upregulated, bcl-2 expression had no obvious change. The animal experiment confirmed knockdown of survivin could inhibit the tumor growth.

CONCLUSIONS

Survivin-specific siRNA can efficiently suppress the expression of survivin, increase apoptosis, inhibit cells proliferation and enhance the chemosensitivity to cisplatin in vitro and in vivo. Suppression of survivin expression helping to reverse drug-resistance may have relationship with downregulation of LRP and upregulation of caspase-3. Anti-tumor strategies based on the inhibition of survivin may be useful in targeting lung adenocarcinomas.

摘要

背景

凋亡抑制蛋白（IAP）家族成员 Survivin 在肿瘤细胞中过度表达，而在终末分化的成人组织中不表达。本研究旨在探讨 Survivin 特异性 siRNA 对 A549/DDP 细胞体外和体内增殖、凋亡及顺铂化疗敏感性的影响，并探讨下调 Survivin 表达逆转肿瘤细胞耐药性的机制。

方法

将 Survivin 特异性 siRNA 转染至 A549/DDP 细胞。采用 RT-PCR 法检测 Survivin 和肺耐药相关蛋白（LRP）mRNA 水平，MTT 法检测 A549/DDP（顺铂）细胞对顺铂的化疗敏感性，流式细胞术（FCM）检测细胞凋亡和细胞周期。Western blot 法检测 Survivin、LRP、cyclin-D1、caspase-3 和 bcl-2 蛋白表达水平。体内实验观察 Survivin siRNA 抑制对裸鼠移植瘤生长的影响。

结果

Survivin 特异性 siRNA 能有效下调 Survivin 表达，使细胞周期阻滞于 G2/M 期，凋亡明显增加。抑制 Survivin 表达可降低顺铂的 IC50 和耐药指数，增强肿瘤细胞对顺铂的敏感性。转染 Survivin 特异性 siRNA 后，LRP 和 cyclin-D1 表达下调，caspase-3 表达上调，bcl-2 表达无明显变化。动物实验证实，下调 Survivin 表达可抑制肿瘤生长。