Data_Sheet_1_Long Noncoding RNA FAM201A Mediates the Radiosensitivity of Esophageal Squamous Cell Cancer by Regulating ATM and mTOR Expression via miR-101.ZIP

Background: The aim of the present study was to identify the potential long non-coding (lnc.)-RNA and its associated molecular mechanisms involved in the regulation of the radiosensitivity of esophageal squamous cell cancer (ESCC) in order to assess whether it could be a biomarker for the prediction of the response to radiotherapy and prognosis in patients with ESCC.
Methods: Microarrays and bioinformatics analysis were utilized to screen the potential lncRNAs associated with radiosensitivity in radiosensitive (n = 3) and radioresistant (n = 3) ESCC tumor tissues. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed in 35 ESCC tumor tissues (20 radiosensitive and 15 radioresistant tissues, respectively) to validate the lncRNA that contributed the most to the radiosensitivity of ESCC (named the candidate lncRNA). MTT, flow cytometry, and western blot assays were conducted to assess the effect of the candidate lncRNA on radiosensitivity in vitro in ECA109/ECA109R ESCC cells. A mouse xenograft model was established to confirm the function of the candidate lncRNA in the radiosensitivity of ESCC in vivo. The putative downstream target genes regulated by the candidate lncRNA were predicted using Starbase 2.0 software and the TargetScan database. The interactions between the candidate lncRNA and the putative downstream target genes were examined by Luciferase reporter assay, and were confirmed by PCR.
Results: A total of 113 aberrantly expressed lncRNAs were identified by microarray analysis, of which family with sequence similarity 201-member A (FAM201A) was identified as the lncRNA that contributed the most to the radiosensitivity of ESCC. FAM201A was upregulated in radioresistant ESCC tumor tissues and had a poorer short-term response to radiotherapy resulting in inferior overall survival. FAM201A knockdown enhanced the radiosensitivity of ECA109/ECA109R cells by upregulating ataxia telangiectasia mutated (ATM) and mammalian target of rapamycin (mTOR) expression via the negative regulation of miR-101 expression. The mouse xenograft model demonstrated that FAM201A knockdown improved the radiosensitivity of ESCC.
Conclusion: The lncRNA FAM201A, which mediated the radiosensitivity of ESCC by regulating ATM and mTOR expression via miR-101 in the present study, may be a potential biomarker for predicting radiosensitivity and patient prognosis, and may be a therapeutic target for enhancing cancer radiosensitivity in ESCC.

背景：本研究旨在鉴定参与调控食管鳞状细胞癌（esophageal squamous cell cancer, ESCC）放射敏感性的潜在长链非编码RNA（long non-coding RNA, lncRNA）及其相关分子机制，以评估其是否可作为ESCC患者放疗响应预测及预后评估的生物标志物。
方法：本研究利用芯片技术与生物信息学分析，在放射敏感（n=3）及放射抵抗（n=3）的ESCC肿瘤组织中筛选与放射敏感性相关的潜在lncRNA。采用逆转录定量聚合酶链反应（reverse transcription-quantitative polymerase chain reaction, RT-qPCR）对35份ESCC肿瘤组织（其中20份为放射敏感组织、15份为放射抵抗组织）进行检测，以验证对ESCC放射敏感性贡献度最高的lncRNA（命名为候选lncRNA）。通过MTT实验、流式细胞术及蛋白质印迹（western blot）实验，在ECA109/ECA109R ESCC细胞中体外评估候选lncRNA对放射敏感性的影响。构建小鼠异种移植瘤模型，在体内验证候选lncRNA对ESCC放射敏感性的调控作用。使用Starbase 2.0软件与TargetScan数据库预测候选lncRNA调控的潜在下游靶基因，并通过荧光素酶报告基因实验检测候选lncRNA与潜在下游靶基因间的相互作用，再经PCR实验予以验证。
结果：芯片分析共鉴定出113个异常表达的lncRNA，其中序列相似性家族201成员A（family with sequence similarity 201-member A, FAM201A）被确定为对ESCC放射敏感性贡献度最高的lncRNA。FAM201A在放射抵抗的ESCC肿瘤组织中呈高表达，且与患者放疗短期应答较差及总生存期更短相关。敲低FAM201A可通过负调控miR-101的表达，上调毛细血管扩张性共济失调突变酶（ataxia telangiectasia mutated, ATM）与雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）的表达，从而增强ECA109/ECA109R细胞的放射敏感性。小鼠异种移植瘤模型实验证实，敲低FAM201A可提升ESCC的放射敏感性。
结论：本研究中，lncRNA FAM201A通过调控miR-101介导ATM与mTOR的表达，进而调控ESCC的放射敏感性，其有望成为预测ESCC放射敏感性及患者预后的潜在生物标志物，同时也可作为提升ESCC癌症放射敏感性的治疗靶点。