肺腺癌转移相关转录本1诱导肺癌HCC827细胞对奥希替尼耐药的作用机制  

Mechanism of MALAT1 induced osimertinib resistance in HCC827 lung cancer cells

作　　者：康小红[1] 高园园 王颖[1] 崔艳慧[1] 赵可雷[1] 寇卫政[1] 苗战会[1] 曹飞 龚亚斌 Kang Xiaohong;Gao Yuanyuan;Wang Ying;Cui Yanhui;Zhao Kelei;Kou Weizheng;Miao Zhanhui;Cao Fei;Gong Yabin(Department of Oncology, the First Affiliated Hospital of Xinxiang Medical I nirersity, Xinxiang 453100, China;Department of Oncology, Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200437,China)

机构地区：[1]河南省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岳阳医院肿瘤科,200437

出　　处：《中华肿瘤杂志》2019年第4期257-262,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(2017-01-07-00-10-E00064);新乡医学院研究生科研创新支持计划(YJSCX201725Y).

摘　　要：目的探讨肺腺癌转移相关转录本1(MALAT1)和奥希替尼对肺癌细胞HCC827增殖和凋亡的影响以及MALAT1介导奥希替尼耐药的机制。方法应用LV-vector和LV-over/MALAT1转染HCC827细胞,建立稳定细胞株HCC827/Vector和HCC827/MALAT1。应用短发夹RNA阴性对照(shRNA-NC)、shRNA人表皮生长因子受体3(shRNA-ERBB3)转染HCC827/MALAT1细胞,应用四甲基偶氮唑蓝(MTT)法检测MALAT1、奥希替尼和shRNA-ERBB3对细胞增殖的影响,应用流式细胞术检测MALAT1、奥希替尼和shRNA-ERBB3对细胞凋亡的影响。应用Westernblot检测MALAT1、奥希替尼和shRNA-ERBB3作用后,肺癌细胞中表皮生长因子受体(EGFR)和ERBB3信号通路相关蛋白的表达。结果MTT结果显示,奥希替尼对HCC827/MALAT1细胞增殖的抑制作用显著降低,半数抑制浓度(IC50)>4000nmol/L;但干扰ERBB3后,HCC827/MALAT1细胞对奥希替尼再次敏感,IC50为(17.27±3.21)nmol/L。细胞凋亡实验显示,10nmol/L奥希替尼干预HCC827/MALAT1细胞后,凋亡率为(8.38±0.92)%;但干扰ERBB3后,奥希替尼能促进HCC827/MALAT1细胞凋亡,凋亡率为(27.17±5.83)%,差异有统计学意义(P<0.01);Westernblot检测结果显示,HCC827/MALAT1细胞中磷酸化ERBB3(p-ERBB3)、p-AKT和p-ERK蛋白明显上调,奥希替尼能抑制磷酸化EGFR(p-EGFR)蛋白的表达,但对p-ERBB3、p-AKT和p-ERK蛋白的表达无影响。敲减ERBB3后,奥希替尼能下调p-EGFR、p-ERBB3、p-AKT和p-ERK蛋白的表达水平。结论MALAT1可能是通过激活ERBB3/PI3K/AKT和ERBB3/MAPK/ERK信号通路介导了奥希替尼耐药。Objective To test the effect of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) and/or osimertinib on the proliferation and apoptosis of HCC827 cells, and explore the potential mechanism of MALAT1 induced resistance to osimertinib. Methods We transfected HCC827 cells with LV-vector or LV-over/MALAT1. Stable transfected cells (HCC827/Vector, HCC827/MALAT1) were selected by adding puromycin. HCC827/MALAT1 cells were further transfected with the shRNA-negative control (NC) or shRNA-human epidermal growth factor receptor 3 (ERBB3) plasmid. The effects of overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib on the proliferation of HCC827 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. Cell apoptosis induced by MALAT1 overexpression, knockdown of ERBB3 and/or osimertinib treatment were analyzed by flow cytometry analysis. The expressions of EGFR and ERBB3 signal pathway related proteins in HCC827 cells treated with overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib treatment were detected by western blot. Results The MTT assay showed that sensitivity to osimertinib of HCC827/MALAT1 cells were significantly repressed. The 50% inhibitive concentration (IC50) of osimertinib >4 000 nmol/L in HCC827/MALAT1 cells. However, knockdown of ERBB3 facilitated the anti-proliferation effect of osimertinib, and the IC50 of osimertinib in shRNA-ERBB3 cells was (17.27±3.21) nmol/L. The results of flow cytometry analysis showed that the apoptotic rate of HCC827/MALAT1 cells induced by 10 nmol/L osimertinib was (8.38±0.92)%, significantly lower than (27.17±5.83)% of knockdown of ERBB3 (P<0.01). Western blotting showed that the expression of p-ERBB3, p-AKT and p-extracellular regulated protein kinases (ERK) in HCC827/MALAT1 cells was markedly up-regulated, while the expression of p-epithelial growth factor receptor (EGFR) was inhibited. The expressions of p-ERBB3, p-AKT and p-ERK were marginally affected by osimertinb. However, osimertini

关 键 词：肺肿瘤 肺腺癌转移相关转录子 表皮生长因子受体 奥希替尼 

分 类 号：R734.2[医药卫生—肿瘤]