EGFR突变肺癌中lncRNA表达谱的生物信息学分析及高表达CR749465的验证

发布时间：2018-03-31 20:25

  本文选题：表皮生长因子受体　切入点：长链非编码RNA　出处：《青岛大学》2017年硕士论文

【摘要】：目的:肺癌是常见的恶性肿瘤之一,随着医学的发展,手术、放化疗、靶向治疗及免疫治疗等治疗模式都取得了巨大的进步,但肺癌仍是全球癌症死亡的首要原因。按照不同的组织学的特点,肺癌大体可以分为两种,小细胞型及非小细胞型,其中大部分属为非小细胞型。研究发现在非小细胞肺癌患者中大约40%-80%能够过度表达EGFR,以EGFR为靶点的治疗模式较野生型和传统化疗能明显改善突变患者的生存,已成为NSCLC治疗的新热点。在庞大的人类基因组当中,绝大多数的基因无法进行转录,而余下的极少数基因才能够进行转录,这些基因当中98%以上的基因属于非编码RNA(non-coding RNA,nc RNA)。按照链的长度大小可将非编码RNA分为短链、中链和长链非编码lncRNA(long non-coding RNA)。lncRNA至少有200个核苷酸,是一种不编码蛋白质的RNA。大量研究显示,lncRNA的异常表达对肿瘤的形成和演进有重要的作用。另外,研究发现lncRNA与肝癌、肺癌、乳腺癌、胃癌、结直肠癌等多种肿瘤关系密切。因此,对EGFR突变肺癌的lncRNA表达谱进行生物信息学方面的研究也为肺癌的研究增添了一个新的切入点。我们的研究目的是利用生物信息学分析EGFR突变肺癌中差异表达的lncRNA,对筛选的lncRNA进行功能分析。方法:从NCBI公共数据平台GEO下载肺癌基因芯片数据GSE31210,共获得127例EGFR突变肺癌和20例正常组织的基因表达谱数据。采用稳健多芯片平均标准化(RMA分析)方法对GSE31210表达谱数据进行标准化预处理,预处理后的结果用微阵列显著性分析(SAM)软件进行分析,找出在EGFR突变肺癌中有显著表达差异的lncRNA。对收集的19例EGFR突变肺癌组织和癌旁组织标本进行组织RNA的提取,经荧光定量PCR验证筛选出的候选基因lncRNA CR749465的差异表达。利用DAVID在线软件分析候选lncRNACR749465相关靶基因,进行GO功能分类及pathway通路分析。结果:以实验设定的标准作为差异基因筛选标准(P≤0.05;|(fold change)|≥2),GSE31210芯片筛选的结果表明,在EGFR突变肺癌组织和正常组织中,lncRNA的表达谱有明显差异,有127个基因出现差异表达。在筛选所得的差异lncRNA127个中,其中上调的有96个,下调的有31个。其中上调变化最大的是CR749465,有3.26倍的表达变化差异。经过荧光定量PCR验证,发现候选基因lncRNACR749465基因表达情况与芯片数据分析结果一致。lncRNA CR749465相关靶基因GO分析显示CR749465相关基因可能与血管生成、信号传导、RAN代谢等有关。Pathway分析显示CR749465相关基因可能主要与神经活性的配体-受体相互作用通路、轴突导向通路、细胞粘附分子通路、Rap1信号通路等有关。结论:EGFR突变肺癌存在明显的lncRNA差异性表达.通过GO功能分析及KEGG通路分析发现,CR749465可能在EGFR突变肺癌的发生和发展中发挥重要作用。
[Abstract]:Objective: lung cancer is one of the common malignant tumors, with the development of medicine, surgery, radiotherapy, chemotherapy, targeted treatment and immunotherapy have made great progress. But lung cancer is still the leading cause of cancer death in the world. According to different histological features, lung cancer can be divided into two types: small cell type and non small cell type. Most of them were non-small cell type. About 40% to 80% of patients with NSCLC were able to overexpression EGFR. Treatment targeting EGFR could significantly improve the survival of mutant patients compared with wild-type and conventional chemotherapy. Has become a new hotspot in NSCLC therapy. In the vast human genome, the vast majority of genes cannot be transcribed, while the few remaining genes can be transcribed. More than 98% of these genes belong to non-coding RNA(non-coding RNAs or nc-RNAs. According to the length of the chain, the non-coding RNA can be divided into short strands, and there are at least 200 nucleotides in the medium-chain and long-chain non-coding lncRNA(long non-coding RNA).lncRNA. A large number of studies have shown that abnormal expression of LncRNA plays an important role in tumor formation and progression. In addition, lncRNA has been found to be associated with liver cancer, lung cancer, breast cancer and gastric cancer. Colorectal cancer and many other tumors are closely related. Therefore, The bioinformatics study of lncRNA expression profile of EGFR mutant lung cancer also provides a new entry point for the study of lung cancer. Our aim is to analyze the differential expression of EGFR RNA in EGFR mutant lung cancer by bioinformatics. Functional analysis of screened lncRNA. Methods: from NCBI common data platform GEO download lung cancer gene chip data GSE31210, a total of 127 cases of EGFR mutation lung cancer and 20 cases of normal tissue gene expression profile data, using robust multi-chip average. The GSE31210 expression profile data were preprocessed by the method of standardized RMA analysis. The results of preconditioning were analyzed by microarray significance analysis software (Sam) to find out the significant difference in the expression of LNC NNA in EGFR mutant lung cancer. The RNA was extracted from 19 samples of EGFR mutant lung cancer and paracancerous tissues. The differentially expressed candidate gene lncRNA CR749465 was screened by fluorescence quantitative PCR. The candidate lncRNACR749465 related target genes were analyzed by DAVID online software. Go functional classification and pathway pathway analysis were carried out. Results: the differential gene screening criteria (P 鈮，

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