Data Sheet 1_Diagnostic value of exosome-derived lncRNA PITPNA-AS1 in lung cancer.pdf

BackgroundLung cancer is one of the most lethal types of cancer, and effective diagnostic biomarkers are required. There is increasing evidences that exosome-secreted lncRNAs could play an important role in lung cancer diagnosis. However, the diagnostic value and molecular mechanism of the key lncRNA PITPNA-AS1 in lung cancer remain unclear.
MethodsqRT-PCR was conducted to determine the levels of exosomal lncRNA PITPNA-AS1 in pleural effusions from lung adenocarcinoma, squamous cell lung carcinoma, and small cell lung cancer patients. Receiver operating characteristic (ROC) curve analyses were used to evaluate the diagnostic accuracy of PITPNA-AS1. Its role in lung cancer development was determined by a series of experiments, including CCK-8, flow cytometry, and transwell assays. RNA pull-down and RNA immunoprecipitation assays were carried out to examine the interaction between PITPNA-AS1 and Fragile X messenger ribonucleoprotein 1 (FMR1).
ResultsWe discovered PITPNA-AS1 in exosomes from lung cancer patients. Its expression was significantly increased in lung cancer patients compared to non-cancer patients, and it was strongly associated with tumor stage, lymph node metastasis, and distant metastasis in all lung cancer subtypes assessed (all p<0.05). ROC curve analyses demonstrated that exosomal PITPNA-AS1 had a high accuracy for differentiating among lung cancer subtypes. Furthermore, PITPNA-AS1 boosted H1299 and A549 cell proliferation, migration, and invasion. Mechanistically, via direct interaction, PITPNA-AS1 increased FMR1 stability by preventing its ubiquitination.
ConclusionsThese results reveal that exosome-derived lncRNA PITPNA-AS1 acts as an oncogene to promote malignant biological behaviors and is a promising diagnostic biomarker in lung cancer.

背景：肺癌是致死率最高的癌症类型之一，亟需有效的诊断生物标志物。越来越多的证据表明，外泌体（exosome）分泌的长链非编码RNA（long non-coding RNA，lncRNA）在肺癌诊断中发挥重要作用。然而，关键长链非编码RNA PITPNA-AS1在肺癌中的诊断价值与分子机制仍未明确。
方法：采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测肺腺癌、鳞状细胞肺癌及小细胞肺癌患者胸膜积液中外泌体长链非编码RNA PITPNA-AS1的表达水平。通过受试者工作特征曲线（Receiver Operating Characteristic，ROC）分析评估PITPNA-AS1的诊断效能。通过细胞计数试剂盒-8（Cell Counting Kit-8，CCK-8）实验、流式细胞术（flow cytometry）、Transwell小室实验等一系列实验探究其在肺癌发生发展中的作用。采用RNA下拉实验与RNA免疫沉淀实验检测PITPNA-AS1与脆性X信使核糖核蛋白1（Fragile X messenger ribonucleoprotein 1，FMR1）的相互作用。
结果：本研究在肺癌患者来源的外泌体中检测到PITPNA-AS1的表达。与非癌症患者相比，肺癌患者体内该RNA的表达水平显著升高，且在所有评估的肺癌亚型中，其表达与肿瘤分期、淋巴结转移及远处转移均显著相关（所有p<0.05）。ROC曲线分析显示，外泌体PITPNA-AS1对区分不同肺癌亚型具有较高的诊断准确度。此外，PITPNA-AS1可促进H1299与A549细胞的增殖、迁移与侵袭。机制研究表明，PITPNA-AS1通过与FMR1直接结合，抑制其泛素化修饰，从而增强FMR1的稳定性。
结论：本研究结果表明，外泌体来源的长链非编码RNA PITPNA-AS1可作为癌基因促进肺癌的恶性生物学行为，是一种极具应用前景的肺癌诊断生物标志物。