任婕;彭芝兰;杨英捷;肖子文;
REN Jie;PENG Zhilan;YANG Yingjie;XIAO Ziwen;Department of Obstrics and Gynecology,Affiliated hospital of Guizhou medical University;Department of Obstetrics and Gynecology,Second Hospital of West China Medical Center of Sichuan University;

• 1:贵州医科大学附院妇产科
• 2:四川大学华西第二医院妇产科

摘要(Abstract)：

目的:探讨利用RNAi沉默cdc2基因逆转人卵巢上皮性癌细胞顺铂耐药的可行性,为卵巢癌顺铂耐药逆转提供一个可选择的靶点。方法:针对cdc2基因的DNA设计具有特异性的SiRNA,经脂质体转染卵巢癌顺铂耐药细胞A2780/DDP,采用逆转录-聚合酶链式反应(RT-PCR)和免疫印迹法检测转染组cdc2基因表达是否抑制;用MTT法测定转染前后A2780/DDP细胞对顺铂敏感性的变化,流式细胞仪检测细胞凋亡率。结果:(1)转染cdc2-SiRNA在RNA及蛋白水平均有效抑制A2780/DDP细胞中cdc2的表达,抑制率分别为78.2%、76.8%;(2)顺铂化疗IC50,A2780/DDP-S(0.66±0.02)较A2780/DDP(5.02±0.05)显著下降(P<0.01);(3)5 mg/L顺铂作用后,A2780/DDP-S与A2780/DDP比较,各时间点细胞生长抑制率明显增加(P<0.05),细胞凋亡率明显增多(P<0.05)。结论:在A2780/DDP细胞中,SiRNA能有效抑制cdc2基因的表达,并能部分恢复其对顺铂化疗的敏感性。
Objective: To investigate the feasibility of RNAi silencing of the cdc2 gene in human ovarian carcinoma cell line A2780 / DDP to reverse the cisplatin resistancein,and provide a novel therapy target for ovarian chemotherapy resistance. Methods: The specific SiRNA was synthesized for cdc2 and transfected into human ovarian carcinoma cisplatin resistance cell line A2780 / DDP by liposome.RT-PCR and cell immunostaining were used to detect the changes of cdc2 mRNA and protein in A2780 / DDP cell. The drug sensitivity to cisplatin was examined by MTT assay. Apoptosis rates were detected by flow cytometry. Results:( 1) The transfection of cdc2-SiRNA could effectively inhibit the expression of cdc2 RNA and protein in A2780 / DDP cells. The inhibition rate was 78. 2% and 76. 8%respectively.( 2) The IC50 of A2780 / DDP-S( 0. 66 ± 0. 02) was significantly decreased compared with A2780 / DDP( 5. 02 + 0. 05).( 3) After treated by cisplatin( 5 mg / L),the cell growth inhibition rate and apoptosis rate of A2780 / DDP-S was significantly increased than that of A2780 / DDP at each time point( P < 0. 05). Conclusion: The SiRNA could effectively block the cdc2 gene expression in A2780 / DDP cell,thereby reversed resistance to cisplatin in some part.

关键词(KeyWords)： 人卵巢癌;RNA干扰;cdc2;顺铂耐药;免疫印迹法;逆转录-聚合酶链式反应
human ovarian carcinoma;RNA interference;cdc2;cisplatin resistance

Abstract:

Keywords:

基金项目(Foundation): 贵州省科技厅资助项目(No:E2009-39)

作者(Author): 任婕;彭芝兰;杨英捷;肖子文;
REN Jie;PENG Zhilan;YANG Yingjie;XIAO Ziwen;Department of Obstrics and Gynecology,Affiliated hospital of Guizhou medical University;Department of Obstetrics and Gynecology,Second Hospital of West China Medical Center of Sichuan University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2015.12.010

参考文献(References)：

• [1]Zhang PN,Zhang PF,Zhou M,et al.Exon 4 deletion variant of epidermal growth factor receptor enhances invasiveness and cisplatin resistance in epithelial ovarian cancer[J].Carcinogenesis,2013(5):2639-2646.
• [2]Huo F,Yu J,Beale P,et al.Combinations of Platinums and Selected Phytochemicals as a Means of Overcoming Resistance in Ovarian Cancer[J].Anticancer Res,2014(6):541-545.
• [3]Zhang X,Huang LN,Zhao YL,et al.Downregulation of miR-130a contributes to cisplatin resistance in ovarian cancer cells by targeting X-linked inhibitor of apoptosis(XIAP)directly[J].Acta Biochim Biophys Sin,2013(5):995-1001.
• [4]DupréA,Buffin E,Roustan C,et al.The phosphorylation of ARPP19 by Greatwall renders the auto-amplification of MPF independently of PKA in Xenopus oocytes[J].Cell Sci,2013(4):3916-3926.
• [5]Tan M,Jing T,Lan KH,et al.Phosphorylation on tyrosine-15 of p34(Cdc2)by Erb B2 inhibits p34(Cdc2)activation and is involved in resistance to taxol-induced apoptosis[J].Mol Cell,2002(5):993-1004.
• [6]蒋铁根,刘建华,郑志娟,等.紫杉醇联合顺铂治疗晚期老年卵巢癌的临床分析[J].肿瘤药学,2004(5):370-373.
• [7]范丽梅,苏静,董慧,等.抑制葡萄糖调节蛋白78表达逆转人卵巢癌细胞顺铂耐药机制的分析[J].中华医学杂志,2013(17):1341-1344.
• [8]Zempolich K,Fuhrman C,Milash B,et al.Changes in gene expression induced by chemoradiation in advanced cervical carcinoma:a microarray study of RTOG C-0128[J].Gynecol Oncol,2008(2):275-279.
• [9]Li X,Wang G,Zhao J,et al.Antiproliferative effect of beta-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase[J].Cell Mol Life Sci,2005(7-8):894-904.
• [10]Gianluca C,Anastasia M,Domenico A,et al.RNAiMediated Silencing of Myc Transcription Inhibits Stemlike Cell Maintenance and Tumorigenicity in Prostate Cancer[J].Cancer Res,2013(2):6816-6827.
• [11]Keita U,Takahiro O,Fumitaka T.RNAi Therapeutics and Applications of MicroRNAs in Cancer Treatment.Jpn[J].Clin Oncol,2013(1):596-607.
• [12]Ley S,Weigert A,Heriche JK,et al.RNAi screen in apoptotic cancer cell-stimulated human macrophages reveals co-regulation of IL-6/IL-10 expression[J].Immunobiology,2013(1):40-51.

文章评论(Comment)：