The relatronship of ERCC1 gene with platinum resistance in epithelial ovarian cancer cells.

doi:10.7504/fk2015030116

Abstract

Abstract:

Abstract： Objective To detect the expression of excision repair cross-complementation group 1 (ERCC1) gene in the cisplatin-resistant human ovarian cancer cell lines SKOV3 / DDP and its parent cell lines SKOV3, in order to explore the significance of ERCC1 gene in ovarian cancer cisplatin resistance and whether the effective ERCC1 gene silencing can effectively reverse the resistance of SKOV3 / DDP cells to DDP. Methods By real-time fluorescent quantitative PCR (RT-PCR) and Western imprinting method,detect the expression of ERCC1 mRNA and protein in SKOV3 / DDP and SKOV3 cell. Construct three artificial short hairpin RNA (shRNA) plasmid expression vector of ERCC1 gene silence, transfect to SKOV3 / DDP cells by slow transfection viral vector method. Detect the level of ERCC1 gene silence,and chose the best silent expression vector by RT-PCR and Western imprinting method. Detect the expression of ERCC1 gene in SKOV3 / DDP cells after stable transfection by RT-PCR and Western imprinting method and detect the resistance of transfection cells to DDP by CCK 8. Results The expression of ERCC1 mRNA and protein in SKOV3 / DDP cells was significantly higher than that of SKOV3, the difference being statistically significant (P< 0.01). The expression of ERCC1 mRNA and protein in SKOV3 / DDP cells was obviously decreased after stable transfection of ERCC1 shRNA, the difference being statistically significant (P<0.01). That suggested the specific ERCC1 gene silence could increase the sensitivity of SKOV3 / DDP cells to cisplat by Cell activity detection (P<0.05). Conclusion The expression of ERCC1 gene in SKOV3 / DDP cells increases significantly.The effective ERCC1 gene silence can effectively reverse the resistance of SKOV3 / DDP cells to cisplatin, increase sensitivity of the ovarian epithelial carcinoma cisplatin resistant cells to cisplatin,and thus provide new targets for the treatment of epithelial ovarian cancer drug resistance.

Key words: ovarian tumors, small interfering RNA, resistance, tumor, ERCC1 gene

摘要：

目的检测切除修复交叉互补1（ERCC1）基因在人卵巢上皮癌顺铂(DDP)耐药细胞株SKOV3／DDP及其亲本细胞株SKOV3中的表达，探讨ERCC1基因在卵巢上皮癌顺铂耐药中的意义以及有效沉默ERCC1基因能否有效逆转SKOV3／DDP细胞对DDP耐药。方法于2013—2014年在南方医科大学采用实时荧光定量PCR(RT-PCR)和Western印迹法检测 ERCC1 mRNA和蛋白在SKOV3／DDP及SKOV3细胞中的表达。人工构建3个靶向沉默ERCC1基因的短发夹状RNA(shRNA)质粒表达载体，慢病毒载体法转染至SKOV3／DDP细胞，RT-PCR和Western印迹法检测ERCC1基因水平并挑选沉默效果最佳的表达载体。RT-PCR和Western印迹法检测稳定转染后SKOV3／DDP细胞中ERCC1基因的表达，CCK-8法检测转染细胞对DDP的耐药性。结果 SKOV3／DDP细胞中ERCC1 mRNA和蛋白的表达量明显高于SKOV3，差异有统计学意义(P<0.01)。稳定转染ERCC1 shRNA后的SKOV3／DDP细胞，ERCC1的mRNA及蛋白的表达量明显下降，差异有统计学意义(P<0.01)。细胞活性检测提示特异性沉默ERCC1基因能增加SKOV3/DDP 细胞对顺铂的敏感性(P<0.05)。结论 ERCC1基因在SKOV3／DDP细胞中的表达明显升高，有效沉默ERCC1 基因能有效逆转SKOV3/DDP细胞对DDP耐药，增加卵巢上皮癌DDP耐药细胞对DDP的敏感性，为卵巢上皮癌耐药的治疗提供新靶点。

关键词: 卵巢肿瘤, 小分子干扰, 抗药性, 肿瘤, ERCC1基因

CLC Number:

R737.3

DU Pei*,WANG Yi-feng, ZHANG Xiao-wei,CHEN Li-quan,GAN Ya-ping.. The relatronship of ERCC1 gene with platinum resistance in epithelial ovarian cancer cells.[J]. Acta Metallurgica Sinica, DOI: 10.7504/fk2015030116.

杜培1,2，王沂峰２，张晓薇3，陈礼全3，淦亚萍3,. ERCC1基因与卵巢上皮癌细胞铂类耐药相关性探讨[J]. 中国实用妇科与产科杂志, DOI: 10.7504/fk2015030116.

References

［1］    柳艳飞，管晓翔，陈龙邦，等.ERCC1与XPD和XPA的遗传多态性对非小细胞肺癌铂类化疗敏感性预测的研究［J］.中华肿瘤防治杂志，2008，15(17)：1285-1288.

［2］   Feng-Ying L, Xiao-Bin R, Xin-You X，et al. Meta-analysis of Excision Repair Cross-complementation Group 1 (ERCC1) Association with Response to Platinum-based Chemotherapy in Ovarian Cancer［J］. Asian Pacific J Cancer Prev, 2013,14,7203-7206.

［3］    赵丹，张伟，李晓，等. 乳腺癌易感基因1,核苷酸切除修复交叉互补基因1, β-微管蛋白Ⅲ和富脯氨酸多肽13基因mRNA的表达与原发性卵巢上皮性癌临床化疗敏感性的关系［J］. 中华肿瘤杂志，2012,34（3）：196-200.

［4］   Mustafa ZM, Ioana B,Mani N,et al.ERCC1 expression as a predictor of resistance to platinumbased chemotherapy in primary ovarian cancer［J］. Anticancer Res,2014,34: 393-400.

［5］    林肖鹰，陈小岩，黄颖,等.切除修复交叉互补基因1在乳腺癌中的表达及意义［J］. 临床肿瘤杂志，2013,12（18）：1096-1099.

［6］    张树才, 王敬慧. 2010年非小细胞肺癌NCCN治疗指南解读［J］. 结核病与胸部肿瘤，2010，5（2）：148-157.

［7］    Jennifer M，Rubatt KM，Darcy,et al. Pre-treatment tumor expression of ERCC1 in women with advanced stage epithelial ovarian cancer is not predictive of clinical outcomes:a gynecologic oncology group study［J］. Gynecol Oncol， 2012, 125: 421-426.

［8］    Steffensen KD, Waldstrøm M, Jeppesen U,et al. Prediction of response to chemotherapy by ERCC1 immunohistochemistry and ERCC1 polymorphism in ovarian cancer［J］. Int J Gynecol Cancer,2008,18:702-710.

［9］    Lin H， Changcllien CC. Management of relapsed/refractory epithelial ovarian cancer: current standards and novel approaches［J］. Taiwan Obstet Gynecol，2007，46：379-388.

［10］   Colombo N, Core M. Treatment of recurrent ovarian cancer relapsing 6-12 months post platinum-based chemotherapy［J］.Crit Rev Oncol Hematol，2007，64：129-138.

［11］    Niedemhofer LJ，Odijk H，Budzowska M，et al.The structure-specific endonuclease Erccl-Xpf is required to resolve DNA interstrand cross-link-induced double-strand bleaks［J］.Mol Cell Biol，2004，24(13)：5776-5787.

［12］    Andrieux LO，Fantrel A，Bessard A，et al.GATA-l is essential in EGF-mediated induction of nucleotide excision repair activity and ERCC l expression through ERK2 in human hepatoma cells ［J］.Cancer Res，2007，67(5)：2114-2123.

［13］    Sidahmed AM,Wilkie B. Endogenous antiviral mechanisms of RNA interference：a comparative biology perspective［J］.Methods Mol Biol,2010,6(23):3-19.

［14］   Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells［J］. Science,2002,296(5567):550-553.

（2014-12-23收稿    2015-01-19修回）

[1]	. [J]. Acta Metallurgica Sinica, 2019, 35(9): 1000-1007.
[2]	CHANG Miao-miao, ZOU Zhong-wen, TAN Shuang.. Expression of GILZ and Ki-67 in epithelial ovarian carcinoma and its clinical implication. [J]. Acta Metallurgica Sinica, 2014, 30(12): 969-972.