鼻咽癌同期放化疗抵抗细胞来源的外泌体lncRNAs表达谱分析

Expression profiling of cell-derived exosomal lncRNAs resistant to concurrent chemoradiotherapy in nasopharyngeal carcinoma

目的筛选出导致鼻咽癌细胞产生放化疗抵抗的关键外泌体长链非编码RNA(lncRNAs)分子。方法体外构建人鼻咽癌细胞同期放化疗模型,采用大剂量同期放化疗连续冲击法诱导建立鼻咽癌放化疗抵抗细胞株,并验证其抵抗性形成。分别提取和鉴定鼻咽癌放化疗抵抗细胞株和各自的母细胞株产生的外泌体,运用生物芯片技术检测外泌体lncRNAs的表达差异。结果经过10次同期放化疗反复处理,成功筛选出放化疗抵抗细胞株CNE-1 CRR、CNE-2 CRR。与母细胞株相比,CNE-1 CRR、CNE-2 CRR有上皮向间质形态转化倾向,细胞克隆形成数目更多且平均致死剂量(D0)、准阈剂量(Dq)、2 Gy照射后细胞存活率(SF2)、细胞存活率数值更高。利用外泌体lncRNAs PCR芯片检测鼻咽癌细胞,相较于其各自母细胞株,CNE-1 CRR外泌体中有18个lncRNAs显著上调和31个lncRNAs显著下调,CNE-2 CRR外泌体中有15个lncRNAs显著上调和38个lncRNAs显著下调,CNE-1 CRR与CNE-2 CRR还具有相似的表达谱。结论CNE-1 CRR、CNE-2 CRR的外泌体中共同存在显著上调和下调的lncRNAs。

Objective To screen the key exosomal long non-coding RNAs(lncRNAs)molecules that cause nasopharyngeal carcinoma cells to develop chemoradiotherapy resistance.Methods In vitro,a model of concurrent chemoradiotherapy for human nasopharyngeal carcinoma cells was constructed,and the continuous shock method of high-dose concurrent chemoradiotherapy was used to induce the establishment of chemoradiotherapy-resistant nasopharyngeal carcinoma cell lines,and its resistance formation was verified.Exosomes produced by chemoradiotherapy-resistant cell lines and respective mother cell lines for nasopharyngeal carcinoma were extracted and identified.Finally,biochip technology was used to detect the differential expression levels of exosomal lncRNAs.Results After 10 repeated treatments of concurrent chemoradiotherapy,CNE-1 CRR and CNE-2 CRR were successfully obtained.Compared with the mother cell lines,CNE-1 CRR and CNE-2 CRR had a tendency to transform from epithelial to interstitial morphology,and the number of cell clones was higher,and the values of average lethal dose(D0),quasi-threshould dose(Dq),survival fraction after 2 Gy irradiation(SF2)and cell survival rate were higher.Nasopharyngeal carcinoma cells were detected by PCR chip of exosomal lncRNAs.Compared with their respective mother cell lines,18 lncRNAs in CNE-1 CRR exosomes were significantly up-regulated and 31 lncRNAs were significantly down-regulated,and 15 lncRNAs were significantly up-regulated and 38 lncRNAs were significantly down-regulated in CNE-2 CRR exosomes.CNE-1 CRR also had similar expression profiles to CNE-2 CRR.Conclusion There are significantly up-regulated and down-regulated lncRNAs in the exosomes of CNE-1 CRR and CNE-2 CRR.