RNA干扰survivin基因提高淋巴瘤细胞对阿霉素的敏感性

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

目的:探讨RNA干扰下调Burkitt淋巴瘤Daudi细胞系凋亡抑制基因survivin的表达对阿霉素敏感性的影响。方法:构建survivin发夹RNA(shRNA)真核表达载体,脂质体介导转染Daudi细胞。多重RT-PCR检测survivin mRNA表达;Western blotting检测Survivin蛋白表达;流式细胞仪(FCM)检测干扰前后细胞凋亡率;MTT法检测干扰前后细胞对化疗药物阿霉素(adriamycin,ADR)敏感性变化。结果:与无功能control-shRNA处理组和PBS处理组比较,转染survivin-shRNA细胞的survivin mRNA和蛋白表达率显著降低,抑制率分别为62.32%和61.88%(P<0.05);FCM结果示转染组细胞凋亡指数(apoptosis index,AI)显著高于两对照组(P<0.05);MTT结果显示,转染组细胞的阿霉素半数抑制浓度(IC50)为(0.25±0.43)μmol/L,显著低于无功能control-shRNA处理组(0.87±0.21)μmol/L和PBS处理组(0.91±0.36)μmol/L,P<0.05;相同剂量ADR对转染细胞的生长抑制率明显高于PBS组和control组。结论:发夹RNA干扰survivin基因可显著提高Daudi细胞凋亡率和对化疗药物阿霉素的敏感性。

AIM ： This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitt＇s lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS ： The survivin - shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT- PCR and Western blotting analysis, respectively. Apeptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin （ADR） before and after transfection was detected by Mrl＇r test. RESULTS ： The mRNA and protein levels of survivin were down- regulated by 62. 32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group （P 〈 0. 05）. Meanwhile, the apeptosis index was significantly increased （19. 10% ±2. 15% ）, compared to that in control group （4. 48%±1.54% ） and PBS group （4. 35%±1.37%, P 〈 0. 05 ）. The 50% inhibition concentration （ IC50 ） of ADM to Daudi cells was significantly decreased （0. 25±20. 43） μmol/L, compared to that in control group （0. 87±20. 21 ） μmol/L and PBS group （0. 91±20. 36） μmol/L, P 〈0. 05. CONCLUSION： Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.