摘要
目的研究EH结构域蛋白2(EHD2)对HER-2阳性乳腺癌细胞曲妥珠单抗(TZB)敏感性的影响及作用机制。方法联合分析GEO、Kaplan-Meier plotter及TCGA数据库,筛选出与乳腺癌细胞TZB敏感性密切相关的基因EHD2。体外培养人乳腺癌细胞BT474,将其分为敏感组、抵抗组及干扰组,敏感组用PBS处理12周后转染PX459-Control载体,抵抗组用0.5、1、2、4、8、16μg·mL^(-1)TZB诱导12周后转染PX459-Control载体,干扰组用0.5、1、2、4、8、16μg·mL^(-1)TZB诱导12周后转染PX459-sgEHD2载体。CCK-8实验检测细胞的TZB半抑制浓度(IC_(50)),实时荧光定量PCR及蛋白质印迹法检测EHD2及其潜在下游基因血小板源性生长因子β受体(PDGFRβ)的表达。结果GEO数据库显示,EHD2在TZB抵抗的乳腺癌细胞中表达显著上调(P<0.05);Kaplan-Meier plotter数据库分析发现,EHD2高表达与HER-2阳性乳腺癌患者预后不良显著相关(P<0.05),与HER-2阴性乳腺癌患者生存时间延长显著相关(P<0.05);TCGA数据库分析显示,乳腺癌组织中EHD2与PDGFRβ表达水平呈正相关(P<0.01)。与敏感组比较,抵抗组与干扰组TZB的IC_(50)显著升高,抵抗组EHD2、PDGFRβ的表达均显著上调,干扰组EHD2表达显著下调,PDGFRβ表达显著上调,差异均有统计学意义(P<0.05);与抵抗组比较,干扰组TZB的IC_(50)显著降低,EHD2、PDGFRβ的表达均显著下调,差异有统计学意义(P<0.05)。结论EHD2可促进HER-2阳性乳腺癌细胞的TZB抵抗,其机制可能与上调PDGFRβ表达有关。
Objective To investigate the effects of EH domain containing 2(EHD2)on the sensitivity of HER-2 positive breast cancer cells to trastuzumab(TZB).Methods The database of GEO,Kaplan-Meier plotter and TCGA were analyzed to screen the gene EHD2 which is closely related to the sensitivity of breast cancer cells to TZB.BT474 cells were divided into sensitive group,resistance group and interference group.The sensitive group was treated with PBS for 12 weeks and then transfected with PX459-Control vector.The resistance group was induced with 0.5,1,2,4,8,16μg·mL^(-1)TZB for 12 weeks and then transfected with PX459-Control vector.The interference group was treated with 0.5,1,2,4,8,16μg·mL^(-1)TZB for 12 weeks and then transfected with PX459-sgEHD2 vector.CCK-8 assay was used to detect the 50%inhibiting concentration(IC_(50))of TZB.Real-time PCR and Western blotting were used to detect the expressions of EHD2 and its potential downstream gene platelet-derived growth factor receptorβ(PDGFRβ).Results The expression of EHD2 in TZB-resistant breast cancer cells was significantly increased from GEO database(P<0.05).The high EHD2 expressions were significantly correlated with poor prognosis of breast cancer patients with positive HER-2 and prolonged survival of breast cancer patients with negative HER-2(P<0.05).TCGA database analysis showed that there was a significant positive correlation between EHD2 and PDGFRβexpression in breast cancer tissues(P<0.01).Compared with the sensitive group,the TZB IC_(50)of resistance group and interference group were significantly increased,the expressions of EHD2 and PDGFRβin resistance group were significantly up-regulated,while the expression of EHD2 in interference group was significantly downregulated,and the expression of PDGFRβin interference group was significantly up-regulated(P<0.05).Compared with the resistance group,the TZB IC_(50)of interference group was significantly decreased,and the expressions of EHD2 and PDGFRβwere significantly down-regulated(P<0.05).Conclusion EHD2 can promote TZB resistance in HER-2 positive breast cancer cells,and its mechanism may be related to the up-regulation of PDGFRβ.
作者
黄莱
李佶刚
陈晓艳
席昕
HUANG Lai;LI Jigang;CHEN Xiaoyan;XI Xin(Department of Pathology,Hunan Cancer Hospital,Changsha,Hunan,410013,China;Electrocardiogram Room,Hunan Cancer Hospital,Changsha,Hunan,410013,China)
出处
《肿瘤药学》
CAS
2021年第6期686-692,共7页
Anti-Tumor Pharmacy
基金
长沙市自然科学基金资助项目(kq2014210)
湖南省卫生健康委员会2019年度科研计划项目(C2019066)。
