肺鳞癌全基因组 lncRNAs 表达研究

摘要

Lung cancer is the leading cause of cancer death worldwide, and squamous carcinoma is the most common histological subtype. Clinical and molecular evidence indicated that lung squamous carcinoma is heterogeneous disease. Long non-coding RNAs (lncRNAs) were noncoding RNAs with more than 200 nucleotide length. They have been found to be involved in a variety of biological processes. Many studies indicated that they were aberrantly expressed in some types of carcinomas. In this study, we tested the hypothesis that some lncRNAs may correlate with lung cancer tumor genesis by detecting genome-scale lncRNAs expressions. 16 lung squamous cell carci-noma patients’paired normal and lung tumor tissues were obtained after surgery. First, extracted total RNA from frozen tissues by Trizol reagent; next, reverse-transcripted the total RNA to cDNA, got cRNA in vitro transcription synthesis, and then purified cRNA by spin columns, cRNA was transcribed into cDNA utilizing a random priming method and cDNA was labeled and hybridized to the Agi-lent human 4×180 K microarray. Processed signal data was obtained from hybridized images using Agilent Feature Extraction. Quantile normalization and differential expression data were performed using the Agilent GeneSpring. Data analyses were performed using R and Bioconductor. With abundant and varied probes accounting 38,361 lncRNAs in our microarray, the number of lncRNAs that expressed at a certain level could be detected is 28,055. From the results we found that there were 3,460 lncRNAs that differentially expressed (≥2 absolute fold-change) in lung squamous cell carcinoma tissues compared with normal tissue, among which 127 lncRNAs differentially expressed in all 16 lung squamous cell carcinoma samples. Our study is the first one to determine genome-wide lncRNAs expression pat-terns in lung squamous cell carcinoma by using microarray. The results indicated that clusters of lncRNAs were significantly differentially expressed. Of all 3,460 differentially expressed lncRNAs, 1901 lncRNAs were up-regulated and 1559 down-regulated. 9/16 lung sq-uamous cell carcinoma patients’up-regulated lncRNAs were more common than down-regulated ones. 7/16 patients down-regulated lncRNAs were more common than up-regulated ones. To predict the functions of lncRNAs, we constructed coding-non-coding gene co-expression networks of lncRNAs and mRNAs, on the basic of correlation cutoff 0.99 and P-value<0.05. We searched lncRNA-mRNA pairs whose distance of genomic locations was less than 10kb. In addition, we blatted the sequences of lncRNA and 3‘UTR of mRNAs to screen the sequence similarity lncRNA-mRNA pairs. We then got 33 lncRNA-mRNA pairs. The mRNAs of pairs were considered the target genes of lncRNAs. From the mRNAs, we found that TERC was likely a useful biomarker for the diagnosis of the non-small cell carcinoma. As the ENST00000363312 and sno-RNA_scaRNA_0_548 also had the similar function, we considered that they were likely useful biomarkers for the diagnosis of the non-small cell carcinoma. We concluded that lncRNAs differentially expressed in lung tumor tissues and normal tissues may exert a partial or key role in lung tumor development, and may provide potential targets for future treatment of lung squamous cell carcinoma.%肺癌是世界范围内癌症死亡的主要原因，而肺鳞癌是肺癌中最常见的组织病理类型，约占原发性肺癌的40～50％。临床和分子机制研究表明肺鳞癌是多因素复杂疾病。长链非编码 RNA（long noncoding RNA, lncRNA）是长度大于200 nt 的非编码 RNA 的统称。最初的研究认为它是转录组的“噪音”，但近十年来的生命科学研究发现，它们参与了多项生物调控过程。有研究表明 lncRNAs 在许多癌症类型中均异常表达。本研究通过全基因组 lncRNAs 表达研究探索 lncRNAs 是否有可能与肺鳞癌的形成相关。我们从中南大学湘雅医院收集了16例肺鳞癌手术病人的肺癌及癌旁组织并保存于液氮中，采用 Trizol法提取组织总 RNA，反转录成 cDNA，体外转录合成 cRNA，使用 RNA 纯化柱纯化 cRNA，以 cRNA 为模板，用随机引物进行反转录，荧光标记反转录产物，最后进行 Agilent human 4×180 K 芯片杂交。使用 Agilent Feature Extraction（v10.7）软件对杂交图片进行分析并提取数据，然后用 Agilent GeneSpring 软件对数据进行归一化和差异分析，使用 R 语言对归一化后的表达数据进行统计分析。此芯片中设计了总共38361个 lncRNAs 探针，我们的数据结果显示，肺鳞癌组织组与正常肺组织组比较，有28055个 lncRNAs 探针的表达有一定水平的改变，其中3460个显著差异表达（倍数变化绝对值≥2），而我们对所有样本对统计发现，127个在所有肺鳞癌组织中与配对正常组织相比，其表达有显著差异。在这3460个 lncRNAs 中，1559个 lncRNAs 表达下调，1901个 lncRNAs 表达上调，绝大部分 lncRNAs 的功能未知，因此我们试图通过构建共表达网络预测lncRNAs 的功能。我们以相关系数0.99和 P<0.05为基础，构建了 lncRNAs 和 mRNAs 的共表达网络，并在此基础上从两个方面对 lncRNAs 可能的靶基因进行预测，分别是顺式距离在10 Kb 内的 lncRNA-mRNA 对和与 mRNA 的3’UTR 的序列比对相似的 lncRNA-mRNA 对，共得到33个 lncRNA-mRNA 对。对这33个 mRNA 进行分析发现，TERC 很可能是非小细胞肺癌的诊断标志物，因此考虑与之对应的两个 lncRNA：ENST00000363312和 sno-RNA_scaRNA_0_548也可能作为非小细胞肺癌的诊断标志物。因此我们得出结论：肺鳞癌组织中差异表达的 lncRNAs 可能参与到肺癌的发展过程中，也可能作为未来肺鳞癌的潜在治疗靶标。