Song Liwei, Qian Gang, Huang Jia, Chen Tianxiang, Yang Yunhai
Shanghai Pulmonary Tumor Medical Center, Shanghai Chest Hospital, Shanghai, China.
Department of Thoracic Surgery, Zhangjiagang Third People's Hospital, Suzhou, China.
Ann Transl Med. 2021 Oct;9(20):1593. doi: 10.21037/atm-21-5186.
AZD9291 resistance is still a challenge in the treatment of non-small cell lung cancer (NSCLC) and fibroblasts in the tumor microenvironment (TME) play a key role in the malignant phenotype of NSCLC. The study aimed to investigate the role of exosomes derived from AZD9291-resistant cells on the phenotypes of lung fibroblasts and the underlying mechanism.
The supernatants and exosomes of wild type and AZD9291-resistant NSCLC (H1975/PC9) cells were collected, and co-cultured with lung fibroblasts (MRC-5 cells) respectively. Transwell and quantitative real-time PCR (qRT-PCR) assays were used to evaluate migration and inflammation levels. Exosomes were collected by ultracentrifugation, and identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blots. Microarray was used to screen dysregulated exosomal lncRNAs from the resistant cells. Candidate lncRNAs were selected by bioinformatical annotation of their target genes and verified by qRT-PCR. The target lncRNA was then selected for further confirmation.
Both the supernatant and exosomes from resistant cells significantly promoted the migration of MRC-5 cells, and the exosomes also upregulated mRNA levels of inflammation cytokines. Microarray identified 159 dysregulated exosomal lncRNAs. Fifteen candidate lncRNAs were selected following the biological roles of their target genes. qRT-PCR validation indicated that lnc-MZT2A-5:1 had the highest fold change. Finally, we found that lnc-MZT2A-5:1 could promote the migration ability and inflammation cytokines expression level of MRC-5 cells.
Our study clarified that lnc-MZT2A-5:1 from AZD9291-resistant NSCLC cell lines could promote the activation of MRC-5 cells, thus to uncover a new mechanism for AZD9291 resistance and provide new potential targets for the treatment of NSCLC.
AZD9291耐药性仍是非小细胞肺癌（NSCLC）治疗中的一项挑战，肿瘤微环境（TME）中的成纤维细胞在NSCLC的恶性表型中起关键作用。本研究旨在探讨AZD9291耐药细胞来源的外泌体对肺成纤维细胞表型的作用及其潜在机制。
收集野生型和AZD9291耐药NSCLC（H1975/PC9）细胞的上清液和外泌体，分别与肺成纤维细胞（MRC-5细胞）共培养。采用Transwell和定量实时PCR（qRT-PCR）检测评估迁移和炎症水平。通过超速离心收集外泌体，并采用纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）和蛋白质免疫印迹法进行鉴定。利用基因芯片筛选耐药细胞中外泌体lncRNA的表达失调情况。通过对候选lncRNA靶基因的生物信息学注释筛选候选lncRNA，并经qRT-PCR验证。然后选择目标lncRNA进行进一步确认。
耐药细胞的上清液和外泌体均显著促进MRC-5细胞的迁移，外泌体还上调炎症细胞因子的mRNA水平。基因芯片鉴定出159个表达失调的外泌体lncRNA。根据其靶基因的生物学作用筛选出15个候选lncRNA。qRT-PCR验证表明lnc-MZT2A-5:1的倍数变化最高。最后，我们发现lnc-MZT2A-5:1可促进MRC-5细胞的迁移能力和炎症细胞因子表达水平。
我们的研究表明，AZD9291耐药NSCLC细胞系来源的lnc-MZT2A-5:1可促进MRC-5细胞的活化，从而揭示AZD9291耐药的新机制，并为NSCLC治疗提供新的潜在靶点。
