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Abstract：
Drug resistance is an urgent problem to be solved in the treatment of non‑small‑cell lung cancer (NSCLC). Osimertinib is a third‑generation EGFR‑tyrosine kinase inhibitor, which can improve the efficacy and quality of life of patients; however, the inevitable resistance after long‑term use of osimertinib often leads to treatment failure. Cell lines are key tools for basic and preclinical studies. At present, few osimertinib‑resistant cell lines (HCC827‑OR and H1975‑OR) have been established. In the present study, osimertinib‑resistant cell lines were established by gradually increasing the drug concentration. Half‑maximal inhibitory concentration (IC50), cell morphology, whole exon sequencing, Cell Counting Kit‑8 assay, EdU staining and flow cytometry were used to evaluate the osimertinib‑resistant cell lines. Western blot analysis was used to detect the expression levels of key proteins involved in osimertinib resistance. The circular RNA (circRNA) expression profile was identified by RNA sequencing (RNA‑seq) analysis of HCC827, HCC827‑OR, H1975 and H1975‑OR cells. Subsequently, the biological roles of differentially expressed circRNAs were explored in in vitro studies. Osimertinib‑resistant cell lines were successfully established via treatment with an increasing concentration of osimertinib. Osimertinib IC50 and proliferation of resistant cells were much higher than those of sensitive cells. Notably, phosphorylated (p)‑AKT and p‑ERK were markedly activated in resistant cells, and the inhibitory effect of osimertinib on p‑AKT and p‑ERK was weaker in resistant cells than that in parental cells. RNA‑seq analysis identified differentially expressed circRNAs in HCC827, HCC827‑OR, H1975 and H1975‑OR cells. The most dysregulated circRNAs (circPDLIM5 and circPPP4R1) were selected for further functional study. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the host genes of differentially expressed circRNAs were associated with \'endocytosis\' and \'regulation of autophagy\'. In conclusion, the present study established osimertinib‑resistant cell lines and revealed that circRNAs may serve as a promising biomarker in NSCLC osimertinib resistance.
摘要：
耐药性是目前非小细胞肺癌(NSCLC)治疗中亟待解决的问题。奥希替尼是第三代EGFR酪氨酸激酶抑制剂，这可以提高患者的疗效和生活质量；然而，长期使用奥希替尼后不可避免的耐药性往往导致治疗失败.细胞系是基础和临床前研究的关键工具。目前,已经建立了少数奥希替尼耐药细胞系(HCC827‑OR和H1975‑OR).在本研究中,通过逐渐增加药物浓度建立奥希替尼耐药细胞系.半最大抑制浓度（IC50），细胞形态学,整个外显子测序，细胞计数试剂盒-8测定，使用EdU染色和流式细胞术评估奥希替尼耐药细胞系。Westernblot分析用于检测奥希替尼耐药相关关键蛋白的表达水平。通过HCC827，HCC827-OR的RNA测序（RNA-seq）分析鉴定了环状RNA（circularRNA）表达谱，H1975和H1975-OR细胞。随后，在体外研究中探索了差异表达的circRNAs的生物学作用。通过增加浓度的奥希替尼治疗，成功建立了奥希替尼耐药细胞系。奥希替尼耐药细胞的IC50和增殖明显高于敏感细胞。值得注意的是,磷酸化（p）-AKT和p-ERK在抗性细胞中被显著激活，在耐药细胞中，奥希替尼对p‑AKT和p‑ERK的抑制作用弱于亲本细胞。RNA‑seq分析确定了HCC827、HCC827‑OR、H1975和H1975-OR细胞。选择最失调的circRNAs(circPDLIM5和circPPP4R1)用于进一步的功能研究。京都基因百科全书和基因组途径分析表明，差异表达circRNAs的宿主基因与“内吞”和“自噬调节”相关。总之,本研究建立了奥希替尼耐药细胞系,并揭示circRNAs可能作为NSCLC奥希替尼耐药的一个有前景的生物标志物.
