Pan Rongtao, Zhou Haiyan
Department of Oncology, Taishan Hospital of Shandong Province, Taian 271000, Shandong, People's Republic of China.
Department of Medical Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, Shandong, People's Republic of China.
Cancer Manag Res. 2020 Jun 8;12:4283-4297. doi: 10.2147/CMAR.S241095. eCollection 2020.
Drug resistance restrains the effect of drug therapy in non-small cell lung cancer (NSCLC). However, the mechanism of the acquisition of drug resistance remains largely unknown. This study aims to investigate the effect of exosomal lncRNA H19 on erlotinib resistance in NSCLC and the underlying mechanism.
HCC827 and A549 cells were continuously grafted into erlotinib-containing culture medium to establish erlotinib-resistant cell lines. The expression of H19 and miR-615-3p was detected by qRT-PCR. The protein levels of MMP2, MMP9, CD9, CD63 and ATG7 were measured by Western blot. Cell viability and proliferation were determined by Cell Counting Kit-8 (CCK-8) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, respectively. Migration and invasion were assessed by transwell assay. Xenograft tumor models were used to investigate the effect of H19 on erlotinib resistance in vivo. Online software and dual-luciferase reporter assay were used to predicate the downstream targets and confirm the targeted relationships.
H19 was upregulated in erlotinib-resistant cells, and knockdown of H19 inhibited cell proliferation, migration and invasion in erlotinib-resistant cells. Extracellular H19 can be packaged into exosomes. Exosomes containing H19 induced erlotinib resistance of sensitive cells, while knockdown of H19 abolished this effect. miR-615-3p was a target of H19 and can bind to ATG7. Exosomal H19 affected erlotinib resistance of erlotinib-resistant NSCLC cells via targeting miR-615-3p to regulate ATG7 expression. In addition, the serum exosomal H19 was upregulated in patients with erlotinib resistance. Furthermore, downregulated H19 decreased the resistance of tumor cells to erlotinib in vivo.
Our study demonstrated that exosomal H19 facilitated erlotinib resistance in NSCLC via miR-615-3p/ATG7 axis, which might provide a potential target for the diagnosis and treatment of NSCLC.
耐药性限制了药物治疗非小细胞肺癌（NSCLC）的效果。然而，获得耐药性的机制在很大程度上仍不清楚。本研究旨在探讨外泌体长链非编码RNA H19对NSCLC中厄洛替尼耐药性的影响及其潜在机制。
将HCC827和A549细胞连续接种到含厄洛替尼的培养基中，建立厄洛替尼耐药细胞系。采用qRT-PCR检测H19和miR-615-3p的表达。通过蛋白质印迹法检测MMP2、MMP9、CD9、CD63和ATG7的蛋白水平。分别采用细胞计数试剂盒-8（CCK-8）和3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四氮唑溴盐（MTT）法测定细胞活力和增殖。通过Transwell实验评估迁移和侵袭能力。采用异种移植肿瘤模型研究H19在体内对厄洛替尼耐药性的影响。使用在线软件和双荧光素酶报告基因实验预测下游靶点并确认靶向关系。
H19在厄洛替尼耐药细胞中上调，敲低H19可抑制厄洛替尼耐药细胞的增殖、迁移和侵袭。细胞外H19可被包装到外泌体中。含H19的外泌体诱导敏感细胞产生厄洛替尼耐药性，而敲低H19可消除这种作用。miR-615-3p是H19的靶点，可与ATG7结合。外泌体H19通过靶向miR-615-3p调节ATG7表达，影响厄洛替尼耐药的NSCLC细胞的厄洛替尼耐药性。此外，厄洛替尼耐药患者血清外泌体H19上调。此外，下调H19可降低体内肿瘤细胞对厄洛替尼的耐药性。
我们的研究表明，外泌体H19通过miR-615-3p/ATG7轴促进NSCLC中的厄洛替尼耐药性，这可能为NSCLC的诊断和治疗提供一个潜在靶点。
