摘要
目的观察顺铂(cDDP)对小分子干扰RNA(siRNA)抑制切除修复交叉互补基因1(ERCC1)基因表达的A549细胞增殖、凋亡影响,并探讨cDDP对siRNA抑制ERCC1基因表达的A549细胞化疗敏感性。方法根据ERCC1基因序列,设计合成3对特异性的siRNA(分别命名为S1、S2、S3),同时合成阴性对照siRNA(命名NC)。取对数生长期的A549细胞,采用Lipofectamine2000脂质体转染法进行S1、S2、S3、S1+S2+S3及NC转染,real-time RT-PCR和Western blotting技术检测ERCC1 mRNA和蛋白表达。结果与NC相比,S1、S2、S3及S1+S2+S3均能下调ERCC1表达,但S1+S2+S3下降最显著。用NC及S1+S2+S3转染A549细胞,24 h后加入0、0.5、1、2、4、8μg/mL的cDDP,采用MTS法计算细胞生存率及cDDP对细胞的半数抑制浓度(IC50)。A549细胞转染后48 h加4μg/mL的cDDP,流式细胞仪检测细胞凋亡率。结果加入0、0.5、1、2、4、8μg/mL的cDDP,NC转染的A549细胞存活率分别为100.00%±3.21%、94.86%±7.14%、89.79%±3.29%、66.67%±5.40%、33.31%±1.79%、19.12%±0.41%,S1+S2+S3转染的A549细胞存活率分别为100.00%±6.53%、71.63%±8.23%、59.33%±0.94%、37.42%±1.57%、19.41%±1.41%、12.75%±0.27%,相同cDDP浓度下A549细胞存活率比较,P均<0.05;S1+S2+S3、NC转染细胞的IC50分别为(1.25±0.11)、(3.09±0.36)μg/mL,二者比较,P<0.01。未加cDDP时,NC与S1+S2+S3转染A549细胞凋亡率分别为8.06%±1.79%、14.96%±0.47%,加入cDDP后,细胞凋亡率分别为24.61%±1.98%、43.90%±3.01%,二者比较,P均<0.05。结论 cDDP能降低siRNA抑制ERCC1基因表达的A549细胞增殖能力,并诱导细胞凋亡;cDDP对siRNA抑制ERCC1基因表达的A549细胞化疗敏感性升高。
A bstract： Objce tive bjective： To investigate the effection of excision repair cross complementation 1 （ ERCC1） downr-egulated by RNA interference on proliferation and apoptosis in lung cancer A 549 cell line.Furthermore, to study the chemotherapy sensitivity of A549 cells with down-regulation ERCC1 to cisplatin.Methods：The negative control interfering RNA（NC） together with three small interfering RNAs （S1, S2 and S3） targeting ERCC1 gene was designed and synthe-sized.The siRNAs （S 1, S2, S3, S1＋S2＋S3 and NC） were transfected to A549 cell with lipofectamine 2000, respective-ly.The mRNA and protein expression levels of ERCC 1 were evaluated by real-time RT-PCR and western blot assay .All three siRNAs（S1, S2 and S3） could decrease the expression of ERCC1, but S1＋S2＋S3 reduced ERCC1 mostly.SiRNAs NC and S1＋S2＋S3 were transfected into A549 cell in 96 well-plot, the concentration of 0, 0.5, 1, 2, 4, 8μg/mL cDDP were added 24 hours later , MTS assay was used to measure cell survival rate and cDDP on the cells of the half inhibitory concentration （IC50）.4 μg/mL cDDP was added after 48 hours of interfering NC and S1＋S2＋S3.The apoptosis rate were detected by flow cytometry .Results：At the concentration of 0, 0.5, 1, 2, 4, 8 μg/mL cDDP, the survival rates of A549 cell with NC were 100% ±3.21%, 94.86% ±7.14%, 89.79% ±3.29%, 66.67% ±5.40%, 33.31% ± 1.79%, 19.12% ±0.41%, respectively;While survival rates of A549 cell with S1＋S2＋S3 were 100% ±6.53%,71.63% ±8.23%, 59.33%？ ±0.94%,？ 37.42%？ ±1.57%, 19.41%？ ±？ 1.41%, 12.75% ±？ 0.27%. The cell survival rate of NC and S1＋S2＋S3 group has significant difference at the same concentration of cDDP （P 〈0. 05）.The IC50 of S1＋S2＋S3and NC were （1.25 ±0.11） and （3.09 ±0.36） μg/mL（P〈0.01）.The cell apoptosis rates of NC and S1＋S2＋S3 were 8.06%±1.79% and 14.96%±0.47% without cDDP（P〈0.05）, but the apoptosis rate were 24.61%±1.98% and 43.90%±3.01% with cDDP（P 〈0.05）.Conclusion： Down-regulation ERCC1 by RNAi can enhance the effects of cDDP on promoting apoptosis and inhibiting proliferation of the A 549 cells.The sensitivity of A549 cell to cisplatin can be enhanced by RNA interfering ERCC 1 gene.
出处
《山东医药》
CAS
2014年第19期4-7,共4页
Shandong Medical Journal
基金
中国博士后科学基金面上资助项目(2012M521588)
关键词
切除修复交叉互补基因1
RNA干扰
顺铂
肺肿瘤
肺癌
excision repair cross-complementation group 1
RNA interference
cisplatin
lung neoplasms
lung carcinoma
