南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 242-250.doi: 10.12122/j.issn.1673-4254.2023.02.12

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Lnc-TMEM132D-AS1高表达明显降低非小细胞肺癌对奥希替尼的敏感性

赵齐林，王 楠，李亚霁，吴庆琛，吴兰香   

  1. 重庆医科大学附属第一医院心胸外科，生命科学研究院，重庆 400016
  • 出版日期:2023-02-20 发布日期:2023-03-16

Lnc-TMEM132D-AS1 overexpression reduces sensitivity of non-small cell lung cancer cells to osimertinib

ZHAO Qilin, WANG Nan, LI Yaji, WU Qingchen, WU Lanxiang   

  1. Department of Cardiothoracic Surgery, First Affiliated Hospital of Chongqing Medical University, Institute of Life Sciences, Chongqing Medical University, Chongqing 400016, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 建立奥希替尼获得性耐药非小细胞肺癌（NSCLC）细胞模型，筛选耐药前后差异表达的长链非编码RNA（lncRNAs），并探讨筛选的lncRNA在奥希替尼获得性耐药中的作用及分子机制。方法 奥希替尼获得性耐药细胞株H1975_OR和HCC827_OR是由奥希替尼敏感的NSCLC细胞株H1975和HCC827（分别命名为H1975 _S和HCC827_ S）建立而来；通过CCK-8、克隆形成及流式凋亡实验检测细胞对奥希替尼的敏感性；RNA测序（RNA-seq）、实时荧光定量PCR（qPCR）技术筛选耐药前后差异表达的lncRNAs；采用CCK-8、克隆形成及Transwell实验检测所筛选的lncRNA在NSCLC细胞奥希替尼获得性耐药中的作用；通过核质分离、生物信息学分析及qPCR技术观察该lncRNA的亚细胞定位，并探索其下游互作分子。结果H1975_OR与HCC827_OR对奥希替尼的耐药指数分别为598.70和428.82（P<0.001）。耐药株的生长增殖与克隆形成能力显著提高，而凋亡率降低（P<0.01）。RNA-seq提示H1975_OR与H1975_S之间以及HCC827_OR与HCC827_S之间有共同差异表达的lncRNAs 34个。qPCR验证发现，lnc-TMEM132D-AS1在耐药性NSCLC细胞株中升高最为显著（P<0.01）。TCGA数据库分析提示，lnc-TMEM132D-AS1在NSCLC组织中的表达水平高于癌旁组织（P<0.001），且与患者预后不良相关（P<0.05）。Lnc-TMEM132D-AS1过表达可提高奥希替尼处理下敏感株的生长增殖、克隆形成及迁移能力（P<0.05），而敲低其表达水平可部分恢复耐药株的奥希替尼敏感性（P<0.01）。lnc-TMEM132D-AS1主要定位在胞质，生物信息学分析提示hsa-miR-766-5p是其潜在靶点，两者表达水平呈负相关（P<0.01）。对hsa-miR-766-5p下游的靶mRNAs分析提示相关基因主要集中于Ras信号通路。结论 Lnc-TMEM132D-AS1在奥希替尼获得性耐药NSCLC细胞株中表达显著上调，明显降低了NSCLC细胞对奥希替尼的敏感性，是NSCLC奥希替尼获得性耐药的潜在生物标志物及治疗靶标。

关键词: 长链非编码RNA；lnc-TMEM132D-AS1；NSCLC；奥希替尼；获得性耐药

Abstract: Objective To screen the differentially expressed long non-coding RNAs (lncRNAs) in non- small cell lung cancer (NSCLC) cells with acquired resistance to osimertinib and explore their roles in drug resistance of the cells. Methods The cell lines H1975_OR and HCC827_OR with acquired osimertinib resistance were derived from their osimertinib-sensitive parental NSCLC cell lines H1975 and HCC827, respectively, and their sensitivity to osimertinib was assessed with CCK-8 assay, clone formation assay and flow cytometry. RNA sequencing (RNA-seq) and real-time quantitative PCR (qPCR) were used to screen the differentially expressed lncRNAs in osimertinib-resistant cells. The role of the identified lncRNA in osimertinib resistance was explored using CCK-8, clone formation and Transwell assays, and its subcellular localization and downstream targets were analyzed by nucleoplasmic separation, bioinformatics analysis and qPCR. Results The resistance index of H1975_OR and HCC827_OR cells to osimertinib was 598.70 and 428.82, respectively (P<0.001), and the two cell lines showed significantly increased proliferation and colony-forming abilities with decreased apoptosis (P<0.01). RNA-seq identified 34 differentially expressed lncRNAs in osimertinib-resistant cells, and among them lnc-TMEM132D- AS1 showed the highest increase of expression after acquired osimertinib resistance (P<0.01). Analysis of the TCGA database suggested that the level of lnc-TMEM132D-AS1 was significantly higher in NSCLC than in adjacent tissues (P<0.001), and its high expression was associated with a poor prognosis of the patients. In osimertinib-sensitive cells, overexpression of Lnc-TMEM132D-AS1 obviously promoted cell proliferation, colony formation and migration (P<0.05), while Lnc-TMEM132D-AS1 knockdown partially restored osimertinib sensitivity of the resistant cells (P<0.01). Lnc-TMEM132D-AS1 was localized mainly in the cytoplasm, and bioinformatics analysis suggested that hsa-miR-766-5p was its candidate target, and their expression levels were inversely correlated. The target mRNAs of hsa-miR-766-5p were mainly enriched in the Ras signaling pathway. Conclusion The expression of lnc-TMEM132D-AS1 is significantly upregulated in NSCLC cells with acquired osimertinib resistance, and may serve as a potential biomarker and therapeutic target for osimertinib-resistant NSCLC.

Key words: long non-coding RNA; lnc-TMEM132D-AS1; non-small cell lung cancer; osimertinib; acquired drug resistance