上皮间质转化在NCI-H1975肺腺癌细胞三代EGFR-TKI奥西替尼获得性耐药中的机制研究

Mechanism of epithelial interstitial transformation in third generation EGFR-TKI Osimertinib acquired drug resistance in NCI-H1975 lung adenocarcinoma cells

目的 探究上皮间质转化(EMT)在NCI-H1975肺腺癌细胞三代表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-TKI)奥西替尼获得性耐药中的机制。方法 选取人肺腺癌细胞株NCI-H1975作为实验细胞,采用体外浓度递增的方式诱导建立第三代EGFR-TKI奥西替尼耐药株NCI-H1975OR。使用CCK8法测定增殖能力,使用AnnexinⅤ-FITC/PI双染法测定凋亡能力,使用划痕实验和Transwell侵袭实验测定迁移及侵袭能力,并使用Western Blot法测定不同细胞株EMT相关分子蛋白表达差异。结果 随着奥西替尼药物浓度的增加,两种细胞株的存活率均下降,且在同一药物浓度下,亲本NCI-H1975较耐药株NCI-H1975OR存活数少,亲本NCI-H1975 IC_(50)为(11.24±1.15)nmol/L,耐药株NCI-H1975OR IC_(50)为(5.73±0.75)nmol/L,差异具有统计学意义(P<0.05)。与NCI-H1975组相比,NCI-H1975OR组具有较高的增殖能力(P<0.05)。划痕实验结果显示,在同一时间节点,NCI-H1975OR组较NCI-H1975组划痕两边距离更短;Transwell侵袭实验显示,在同一时间节点,NCI-H1975OR组穿过小室的细胞较NCI-H1975组多。NCI-H1975OR组Vimentin、N-cadherin、Snail、Twist等蛋白表达水平较NCI-H1975组高(P<0.05),E-cadherin蛋白表达水平较NCI-H1975组低(P<0.05)。NF-κB、Wnt/β-catenin等信号通路的关键因子在NCI-H1975OR组中的表达水平较NCI-H1975组高(P<0.05),AKT信号通路的关键因子NCI-H1975OR组中的表达水平较NCI-H1975组低(P<0.05)。结论 EMT可能参与了NCI-H1975肺腺癌细胞三代EGFR-TKI奥西替尼获得性耐药的过程,该机制可能与AKT、NF-κB、Wnt/β-catenin等信号通路的调控有关。

Objective To investigate the mechanism of epithelial mesenchymal transition(EMT)in the acquisition of resistance to Osimertinib,a three representative epidermal growth factor receptor tyrosine kinase inhibitor(EGFR TKI),in NCI-H1975 lung adenocarcinoma cells.Methods The human lung adenocarcinoma cell line NCI-H1975 was selected as the experimental cell line,and the third-generation EGFR-TKI Osimertinib resistant strain NCI-H1975OR was induced by increasing concentration in vitro.The proliferation ability was measured using the CCK8 method,the apoptotic ability was measured using the Annexin V-FITC/PI double staining method,and the migration and invasion ability were measured using scratch and Transwell invasion experiments,and Western blot method was used to determine the differential expression of EMT related molecular proteins among different cell lines.Results With the increase of osimertinib concentration,the survival rate of both cell lines decreased,and at the same drug concentration,the survival number of parent NCI-H1975 was less than that of drug-resistant strain NCI-H1975OR.The parent NCI-H1975 IC_(50) was(11.24±1.15)nmol/L,the drug-resistant strain NCI-H1975OR IC_(50) was(5.73±0.75)nmol/L,with a statistically significant difference(P<0.05).Compared with the NCI-H1975 group,the NCI-H1975OR group had higher proliferation ability(P<0.05).The scratch experiment results showed that at the same time node,the NCI-H1975OR group had a shorter distance on both sides of the scratch compared to the NCI-H1975 group.The Transwell invasion experiment showed that at the same time node,the NCI-H1975OR group had more cells passing through the compartments than the NCI-H1975 group.The protein expression levels of Vimentin,N-cadherin,Snail,and Twist in the NCI-H1975OR group were higher than those in the NCI-H1975 group(P<0.05),while the protein expression levels of E-cadherin were lower than those in the NCI-H1975 group(P<0.05).The expression level of key factors of NCI-κB and Wnt/β-catenin signaling pathway was higher in NCI-H1975OR group than in the NCI-H1975 group(P<0.05),and the expression level of key factors of AKT signaling pathway was lower in the NCI-H1975OR group than in the NCI-H1975 group(P<0.05).Conclusion EMT may be involved in the process of third-generation EGFR-TKI Osimertinib acquired resistance in NCI-H1975 lung adenocarcinoma cells,which may be related to the regulation of AKT,NF-κB,Wnt/β-catenin signaling pathways.