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  

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

作　　者：赵齐林 王楠 李亚霁 吴庆琛[1] 吴兰香[2] ZHAO Qilin;WANG Nan;LI Yaji;WU Qingchen;WU Lanxiang(Department of Cardiothoracic Surgery,First Affiliated Hospital of Chongqing Medical University,Chongqing Medical University,Chongqing 400016,China;Institute of Life Sciences,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院心胸外科,重庆400016 [2]重庆医科大学生命科学研究院,重庆400016

出　　处：《南方医科大学学报》2023年第2期242-250,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(82073938);重庆市自然科学基金(cstc2020jcyj-msxmX0246)。

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。q PCR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。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 lncTMEM132D-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 hs

关 键 词：长链非编码RNA lnc-TMEM132D-AS1 NSCLC 奥希替尼 获得性耐药 

分 类 号：R734.2[医药卫生—肿瘤]