为探索与表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）耐药相关的分子机制，以及潜在的治疗靶点和策略。本研究利用自噬和Beclin 1调节子1（Ambra1）短发夹核糖核酸（shRNA）慢病毒载体和Ambra1过表达质粒构建的质粒克隆脱氧核糖核酸（pcDNA）3.1载体，分别用于人类肺腺癌厄洛替尼耐药细胞株（PC9/ER）中下调和上调Ambra1表达，并筛选稳定转基因细胞株。测量这些细胞株中艾乐替尼的IC50，确定它们的耐药状态。实时定量逆转录聚合酶链式反应（qRT-PCR）用于测量抗药相关基因如多药耐药蛋白1（MDR1）、多药耐药相关蛋白1（MRP1）和肺抗药相关蛋白（LRP）的信使核糖核酸（mRNA）表达。进行Western印迹分析自噬相关基因Beclin 1、LC3II/I和p62的蛋白表达。每个稳定转基因细胞株在裸鼠皮下形成肿瘤；随后将携带PC9/ER细胞和shAmbra1-PC9/ER细胞的皮下肿瘤的裸鼠分别用雷帕霉素（RAPA）和氯喹（CQ）进行治疗。用qRT-PCR检测每个肿瘤组织样品中MDR1、MRP1和LRP的mRNA表达。通过Western印迹分析AMPK、磷酸化AMPK（p-AMPK）、Forkhead box O3（FoxO3a）和磷酸化Forkhead box O3（p-FoxO3a）在AMPK/FoxO3a信号通路中的蛋白表达。qRT-PCR结果显示EGFR-TKI耐药细胞中Ambra1水平增加。Ambra1的过表达加剧了这种情况，在抑制后减少。此外，Ambra1上调了耐药基因mRNA的表达以及自噬相关蛋白的表达。用RAPA处理的shAmbra1-PC9/ER细胞的皮下肿瘤导致耐药基因表达增加，同时p-AMPK减少，p-FoxO3a增加。研究结果显示，模型组中的Beclin-1/β-actin、p62/β-actin和LC3II/I均与对照组相比显著增加（P＜0.05）。与模型组相比，pcDNA-Ambra1组的Beclin-1/β-actin、p62/β-actin和LC3II/I均显著增加（P＜0.05）。与模型组相比，shAmbra1组的Beclin-1/β-actin、p62/β-actin和LC3II/I均显著减少（P＜0.05）。因此，这些数据表明Ambra1促进细胞自噬。用CQ处理的shAmbra1-PC9/ER细胞的皮下肿瘤导致耐药基因表达减少，同时p-AMPK增加，p-FoxO3a减少。本研究结果揭示了Ambra1介导的自噬通过AMPK/FoxO3a信号通路调节NSCLC中的EGFR-TKI耐药。

To explore the molecular mechanisms related to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) resistance, along with potential therapeutic targets and strategies. The autophagy and Beclin 1 regulator 1 (Ambra1) short hairpin ribonucleic acid (shRNA) lentivirus vector and Ambra1 overexpression plasmid, constructed with a plasmid cloning deoxyribonucleic acid (pcDNA) 3.1 vector, were used to down-regulate and up-regulate Ambra1 expression in the human lung adenocarcinoma erlotinib-resistant cell line (PC9/ER), respectively, as well as to screen stable transgenic cell lines. The IC50 of Erlotinib in these cell lines were measured to determine their resistance status. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure messenger ribonucleic acid (mRNA) expression of resistance-related genes like multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung drug-resistant-related protein (LRP). Western blot was performed to analyze the protein expressions of the autophagy-related genes Beclin 1, LC3II/I, and p62. Each stable transgenic line formed a tumor under the skin in nude mice; the mice with subcutaneous tumorigenesis of PC9/ER cells and shAmbra1-PC9/ER cells were subsequently treated with rapamycin (RAPA) and chloroquine (CQ), respectively. The mRNA expressions of MDR1, MRP1, and LRP in each tumor tissue sample were detected by qRT-PCR. The protein expressions of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated-AMPK (p-AMPK), forkhead box O3 (FoxO3a), and phosphorylated forkhead box O3 (p-FoxO3a) in the AMPK/FoxO3a signaling pathway were analyzed via Western blot. The qRT-PCR result revealed that the level of Ambra1 in EGFR-TKI-resistant cells had increased. This was further exacerbated by the overexpression of Ambra1 and was reduced after its inhibition. Additionally, Ambra1 upregulated the mRNA expression of drug-resistant genes and the expression of autophagy-related proteins. Subcutaneous tumorigenesis of RAPA-treated shAmbra1-PC9/ER cells resulted in increased expression of drug resistance-related genes and a concomitant decrease in p-AMPK and increase in p-FoxO3a. The results revealed that Beclin-1/β-actin, p62/β-actin, and LC3II/I in the model group were all significantly increased compared to the control group, with P<0.05. Compared to the model group, Beclin-1/β-actin, p62/β-actin, and LC3II/I were all significantly higher in the pcDNA-Ambra1 group, with P<0.05. Compared to the model group, Beclin-1/β-actin, p62/β-actin, and LC3II/I were all significantly decreased in the shAmbra1 group, with P<0.05. Thus, these data suggest that Ambra1 promotes cellular autophagy. In addition, subcutaneous tumorigenesis of CQ-treated shAmbra1-PC9/ER cells resulted in reduced expression of drug resistance-related genes, and a concomitant increase in p-AMPK and decrease in p-FoxO3a. The results of this study revealed that Ambra1-mediated autophagy regulated EGFR-TKI resistance in NSCLC, most probably through the AMPK/FoxO3a signaling pathway.