赖氨酸羟化酶2诱导肺癌HCC827细胞对奥希替尼耐药的机制研究  

Mechanism of PLOD2 induced osimertinib resistance in non-small cell lung cancer HCC827 cells

作　　者：康小红[1] 王珂 王颖[1] 赵红珂 张娇 赵可雷[1] 苗战会[1] 徐振晔[2] 曹飞 龚亚斌[3] Kang Xiaohong;Wang Ke;Wang Ying;Zhao Hongke;Zhang Jiao;Zhao Kelei;Miao Zhanhui;Xu Zhenye;Cao Fei;Gong Yabin(Department of Oncology,the first Affiliated Hospital of Xinxiang Medical University,Xinxiang 453100,China;Department of Oncology,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Department of Oncology,Yueyang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200437,China)

机构地区：[1]新乡医学院第一附属医院肿瘤科,453100 [2]上海中医药大学附属龙华医院肿瘤科,200032 [3]上海中医药大学附属岳阳中西医结合医院肿瘤科,200437

出　　处：《中华肿瘤杂志》2020年第3期210-215,共6页Chinese Journal of Oncology

基　　金：国家自然科学基金(81503414、81874392);上海市教委科研创新计划项目(2017-01-07-00-10-E00064)。

摘　　要：目的探讨奥希替尼对过表达赖氨酸羟化酶2(PLOD2)的HCC827细胞增殖、迁移和侵袭的影响及其PLOD2诱导奥希替尼耐药的作用机制。方法应用LV-vector和LV-over/PLOD2慢病毒载体转染肺癌HCC827细胞,应用实时荧光定量聚合酶链反应(qRT-PCR)和Western blot法检测PLOD2的表达情况,应用四甲基偶氮唑蓝(MTT)法检测奥希替尼的抗细胞增殖能力,应用细胞划痕实验和Transwell实验检测肿瘤细胞的迁移和侵袭能力,应用免疫荧光法检测E-cadherin和vimentin的表达,应用Western blot法检测上皮间质转化(EMT)、FAK-PI3K/AKT和MAPK信号通路相关蛋白的表达。结果MTT检测结果显示,过表达PLOD2的HCC827-PLOD2细胞对奥希替尼的敏感性降低,半数抑制浓度>1000 nmol/L,耐药指数>100。细胞划痕实验结果显示,HCC827-PLOD2细胞迁移的距离为HCC827-vector细胞的(2.13±0.21)倍。Transwell结果显示,HCC827-PLOD2细胞的穿膜细胞数为(212.78±10.43)个,与对照组HCC827-vector细胞[(101.32±12.52)个]比较,差异有统计学意义(P<0.01)。免疫荧光检测结果显示,与HCC827-vector细胞比较,HCC827-PLOD2细胞中E-cadherin的表达下调,vimentin的表达上调;奥希替尼可下调HCC827-vector细胞中vimentin的表达,上调E-cadherin蛋白的表达,但对HCC827-PLOD2细胞中E-cadherin和vimentin的表达无明显调控作用。Western blot结果显示,过表达PLOD2可下调E-cadherin蛋白的表达,上调vimentin蛋白的表达,奥希替尼可抑制HCC827-PLOD2细胞中磷酸化表皮生长因子受体的表达,但对PLOD2、p-FAK、p-AKT、p-ERK和vimentin等蛋白的表达无明显抑制作用,对E-cadherin蛋白的表达亦无上调作用。结论PLOD2可能通过促进EMT的表达,激活FAK-PI3K/AKT及MAPK信号通路诱导奥希替尼耐药。Objective To investigate the effects of osimertinib on proliferation,migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2(PLOD2)overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance.Methods We transfected HCC827 cells with LV-vector and LV-over/PLOD2.The expression of PLOD2 was detected by quantitative real time polymerase chain reaction(qRT-PCR)and western blotting.The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide(MTT)assay.The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays.The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence(IF).The expressions of epithelial-mesenchymal transition(EMT),FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting.Results The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib.The 50%inhibitory concentration(IC50)and resistance index of osimertinib for HCC827-PLOD2 cells was over 1000 nmol/L and over 100,respectively.The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about(2.13±0.21)fold changes as that of HCC827-vector cells.The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were(212.78±10.43),significantly higher than(101.32±12.52)of HCC827-vector cells(P<0.01).The result of IF showed that compared with HCC827-vector cells,the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells.Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells.The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level.Osimertinib inhibited the exp

关 键 词：肺肿瘤 赖氨酸羟化酶2 奥希替尼耐药 上皮间质转化 黏着斑激酶通路 

分 类 号：R734[医药卫生—肿瘤]