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 osimertinibresistant NSCLC.
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）。LncTMEM132D-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。
Keywords: acquired drug resistance; lnc-TMEM132D-AS1; long non-coding RNA; non-small cell lung cancer; osimertinib.