摘要
目的研究趋化因子配体23(CCL23)及趋化因子受体1(CCR1)与人上皮性卵巢癌细胞顺铂耐药的关系。方法 qRT-PCR法检测人上皮性卵巢癌顺铂耐药细胞C13K及其敏感细胞OV2008中CCL23及CCR1表达;慢病毒转染使OV2008细胞过表达CCL23,嘌呤霉素筛选阳性克隆,倒置荧光显微镜及qRT-PCR观察和检测慢病毒稳定转染效果;BX471(CCR1受体拮抗剂)干预,平板克隆及CCK-8法分别检测细胞增殖活力及顺铂半数抑制浓度(IC50);Western blot检测细胞CCL23、CCR1基因及耐药蛋白YAP表达。结果 qRT-PCR结果显示,C13K中CCL23及CCR1表达均高于OV2008(P<0.01);荧光显微镜下慢病毒转染OV2008细胞满视野绿色荧光表达,CCL23过表达组细胞CCL23 mRNA水平明显上升(P<0.001),成功建立OV2008细胞CCL23稳定表达株,CCL23过表达组细胞CCR1 mRNA表达却无改变;CCL23过表达后OV2008细胞增殖加速,对顺铂耐受性增加,相对于空白对照组及慢病毒空载体组细胞差异有统计学意义(P<0.01);BX471可抑制细胞CCR1及CCL23表达,同时降低细胞增殖活性,增加细胞对顺铂敏感性,抑制细胞中耐药蛋白YAP表达。结论 CCL23/CCR1趋化因子受体轴参与人上皮性卵巢癌细胞顺铂耐药,其机制可能与CCL23/CCR1调控下游效应因子YAP表达有关。
Objective To investigate the role of CCL23 and CCR1 in human epithelial ovarian cancer cells cisplatin resistance. Methods The expression of CCL23 and CCR1 in human epithelial ovarian cancer cisplatin resistant cells C13 K and cisplatin sensitive cells OV2008 was detected by Real-time quantitative PCR(qRT-PCR). CCL23 over expression vector was constructed in OV2008 cells and the positive clones were selected by puromycin. The transfection efficiency was observed by fluorescence microscopy and qRT-PCR. After treated with BX471(CCR1 antagonist), cell proliferation and the half inhibitory concentration of cisplatin(IC50)were assessed by plate clone formation assay and CCK-8 assay respectively. The protein expression of CCL23, CCR1 and YAP(yes-associated protein) was detected by Western blot. Results qRT-PCR showed that the expression of CCL23 and CCR1 in C13 K cells was higher than that in OV2008 cells(P<0.01). Fluorescence microscopy showed that recombinant OV2008 cells were full of green fluorescence. The mRNA level of CCL23 in CCL23 overexpressing cells significantly increased(P<0.001) which proved that the CCL23 gene over expression of OV2008 cells was successfully constructed, but the mRNA level of CCR1 had no significant change(P>0.05). Plate clone formation assay and CCK-8 assay showed that the proliferation ability and cisplatin resistance of OV2008 cell line overexpressing CCL23 increased compared to the empty vector of OV2008 cell line and normal OV2008 cell line(P<0.01). BX471 inhibited the expression of CCL23 and CCR1, meanwhile, downregulated the expression of YAP. Cell proliferation was inhibited and cisplatin sensitivity increased after treated with BX471. Conclusion CCR1/CCL23 axis is involved in EOC cells cisplatin resistance and its mechanism may be related to the regulation of downstream effector YAP by CCL23 and CCR1.
作者
孙磊
张英
朱颍
李泽莲
颜士杰
肖兰
Sun Lei;Zhang Ying;Zhu Ying(Dept of Gynaecology and Obstetrics,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)
出处
《安徽医科大学学报》
CAS
北大核心
2021年第2期272-276,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学青年基金(编号:81603138)
安徽省中央引导地方科技发展专项项目(编号:2017070802D149)
安徽省高校优秀拔尖人才培育项目(编号:gxgwfx2019006)。
