沉默KDM5A基因对卵巢上皮癌顺铂耐药细胞SKOV3/DDP耐药性的影响  

Effect of silencing KDM5A gene on drug resistance of cisplatin-resistant ovarian cancer cell line SKOV3/DDP

作　　者：冯同富[1] 刘玉兰[1] 王波[2] 李卉 杜欣[1] 王燕[1] 李力[3] Feng Tongfu;Liu Yulan;Wang Bo(Department of Gynecology,Maternal and Child Health Hospital of Hubei Province,Wuhan 430070;Clinical Laboratory,Maternal and Child Health Hospital of Hubei Province,Wuhan 430070)

机构地区：[1]湖北省妇幼保健院妇科,武汉430070 [2]湖北省妇幼保健院检验科,武汉430070 [3]广西医科大学区域性高发肿瘤早期防治研究教育部重点实验室,南宁530021

出　　处：《现代妇产科进展》2020年第12期886-891,共6页Progress in Obstetrics and Gynecology

基　　金：湖北省自然科学基金知识创新专项(No:2018CFC840);湖北省卫计委联合基金(No:WJ2018H0158);武汉市中青年医学骨干人培养工程(No.[2018]116)。

摘　　要：目的:探讨沉默KDM5A基因表达对卵巢上皮癌顺铂耐药细胞SKOV3/DDP耐药性的影响及其机制。方法:选择高表达KDM5A的SKOV3/DDP细胞,设计KDM5A基因靶向的发夹状shRNA,筛选出最佳shRNA沉默片段,构建携带有目的基因的重组慢病毒干扰载体并感染SKOV3/DDP细胞,通过嘌呤霉素筛选,RT-PCR和Western blot法验证,建立稳定干扰KDM5A表达的细胞株SKOV3/DDP-KDM5Ai。将实验分为3组,即KDM5A沉默组(SKOV3/DDP-KDM5Ai)、阴性对照组(SKOV3/DDP-NC)和空白对照组(SKOV3/DDP),流式细胞仪检测细胞的周期比例及凋亡情况;CCK-8法检测细胞的IC 50;高效液相色谱分析法检测在不同质量浓度(7.5μg/ml和15μg/ml)顺铂作用48h后细胞内的药物浓度。RT-PCR技术和Western blot法细胞中β-catenin、P-gp及COX-2的mRNA和蛋白表达水平。结果:成功建立KDM5A基因表达沉默的SKOV3/DDP细胞株。SKOV3/DDP-KDM5Ai组细胞凋亡率增高,G 0/G 1期细胞比例明显增高,而S期和G 2期细胞比例明显下降,与SKOV3/DDP-NC组及SKOV3/DDP组比较,差异均有统计学意义(P<0.05)。SKOV3/DDP-KDM5Ai组细胞、SKOV3/DDP-NC组细胞及SKOV3/DDP组细胞对顺铂的IC 50分别为(17.5847±1.1482)μg/ml、(27.8587±0.8876)μg/ml、(28.1267±0.7308)μg/ml,差异有统计学意义(P<0.05)。SKOV3/DDP-KDM5Ai组在不同顺铂质量浓度(7.5μg/ml和15μg/ml)下细胞内顺铂浓度均明显高于SKOV3/DDP-NC组及SKOV3/DDP组(F=5.14,P<0.01)。RT-PCR技术和Western blot法显示,SKOV3/DDP-KDM5Ai组的β-catenin、P-gp及COX-2的mRNA和蛋白表达水平明显低于SKOV3/DDP-NC组及SKOV3/DDP组(P<0.05)。结论:KDM5A基因在顺铂耐药卵巢癌细胞中高表达,其沉默后可在体外降低顺铂耐药的卵巢癌SKOV3/DDP细胞的耐药性,其作用机制可能与Wnt/β-catenin信号通路有关。Objective:To investigate the effect of silencing KDM5A gene expression on cisplatin-resistant ovarian cancer cell line SKOV3/DDP and its mechanism.Methods:Selected SKOV3/DDP cells with high level of KDM5A expression,designed hairpin shRNA targeting KDM5A gene,screened the best shRNA silencing fragment,constructed recombinant lentivirus interference vector with target gene and then infected SKOV3/DDP cells.Through puromycin screening,RT-PCR and Western blot methods,established the cell line SKOV3/DDP-KDM5Ai which stably interfered with the expression of KDM5A.The experiment was divided into three groups:KDM5A silence group(SKOV3/DDP-KDM5Ai),negative control group(SKOV3/DDP-NC)and blank control group(SKOV3/DDP).The cell cycle proportion and apoptosis of the three groups were detected by flow cytometry,and the IC 50 of the three groups were detected by CCK-8.The intracellular drug concentrations of three groups of cells treated with cisplatin at different concentrations(7.5μg/ml and 15μg/ml)for 48h were detected by high performance liquid chromatography(HPLC).The expression levels of mRNA and protein ofβ-catenin,P-gp and COX-2 in the three groups were detected by RT-PCR and Western blot.Results:A SKOV3/DDP cell line with silencing KDM5A gene expression was successfully established.In SKOV3/DDP-KDM5Ai group,the apoptosis rate and the proportion of cells in G 0/G 1 phase increased significantly,while the proportion of cells in S phase and G 2 phase decreased significantly,which were significantly higher than those in SKOV3/DDP-NC and SKOV3/DDP group,respectively(P<0.05).The IC 50 of SKOV3/DDP-KDM5Ai cells,SKOV3/DDP-NC cells and SKOV3/DDP cells to cisplatin were(17.5847±1.1482)μg/ml,(27.8587±0.8876)μg/ml and(28.1267±0.7308)μg/ml,respectively(P<0.05).The intracellular concentration of cisplatin in SKOV3/DDP-KDM5Ai group was significantly higher than that in SKOV3/DDP-NC group and SKOV3/DDP group at different cisplatin concentrations(7.5μg/ml,15μg/ml)(F=5.14,P<0.01).The mRNA and protein expression levels ofβ-

关 键 词：卵巢肿瘤 细胞系 肿瘤 顺铂 化疗 耐药 

分 类 号：R737.33[医药卫生—肿瘤]