顺铂对 siRNA 抑制 ERCC1基因表达的A549细胞增殖、凋亡影响

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。结果与NC相比， S1、S2、S3及S1＋S2＋S3均能下调ERCC1表达，但S1＋S2＋S3下降最为显著。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、8μgm/L 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100．0％±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A 549的细胞凋亡率分别为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细胞增殖能力及IC50，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.