长链非编码RNA PVT1和NEAT1在食管鳞状细胞癌中的表达及意义

发布时间：2018-04-23 10:43

本文选题：长链非编码RNA + PVT1　；参考：《青岛大学》2017年硕士论文

【摘要】：背景及目的食管癌是一种常见的恶性肿瘤,其发病率和致死率在中国分别列第六位和第八位,食管癌的发病往往是由不良的饮食习惯所造成的。它有两种病理学类型:食管鳞状细胞癌和食管腺癌。其中食管鳞癌比较常见。食管癌通常由于患者吞咽困难等不适症状,行胃镜检查时发现,而此时部分患者已处于晚期。目前食管癌的主要治疗手段还是手术,手术不仅会给患者带来痛苦,也可能产生术后吻合口瘘等并发症。因此,早期发现食管癌并可通过分子机制利用药物治疗食管癌成为一种迫切的需求。长链非编码RNA是一类长约200个核苷酸长度的RNA序列,它虽然没有编码蛋白质的作用,但其在胚胎发育,细胞增殖、分化以及调控基因表达方面发挥重要的调节作用。因此推测其在癌症的发生和发展过程中也起到重要的调控作用。PVT1是原癌LncRNA之一,约300bp长,距我们所熟知的MYC基因仅有57kb,由于其特殊的位置关系,推测两者可能发挥协调作用调控肿瘤的发生发展。NEAT1是一种长链非编码RNA,位于人类第1号染色体上,与免疫应答,基因表达调控等生物学过程相关,此前已有相关研究证实NEAT1在神经胶质瘤、白血病、乳腺癌、喉癌等多种肿瘤中表达上调。目前,对上述两个LncRNA的研究主要集中与其他肿瘤,与食管癌的关系鲜有报道,因此本文旨在通过检测长链非编码RNA PVT1和NEAT1在食管鳞状细胞癌患者肿瘤组织和正常组织的差异,并分析其表达含量和食管癌分期及预后的关系,探讨其作为预测食管癌预后和治疗的新的分子标志物。方法1长链非编码RNA PVT1和NEAT1在食管鳞状细胞癌肿瘤组织和配对正常组织的差异性表达研究。1.1收集2015年6月至2016年4月青岛大学医学院附属医院胸外科54例食管鳞状细胞癌患者的正常组织(距肿瘤5cm以上)和肿瘤组织,所有患者术前均未接受放化疗,术后病理确诊为食管鳞状细胞癌,将切除的新鲜癌组织迅速置于含有RNA保护液的EP管中,并于半小时内放置于-80℃的超低温冰箱冻存。1.2 Trizol法分别提取食管鳞状细胞癌肿瘤组织和正常组织中RNA,以微量分光光度计检测所提取RNA的纯度和浓度,取适宜纯度(1.80-2.0)之间的RNA用作下一步实验,不符合要求的重新提取直至符合要求。分别对LncRNA PVT1和LncRNA NEAT1的反转录的上、下游设计引物,利用NCBI网站中的gene数据库查找出lnc RNA PVT1和lnc RNA NEAT1的序列全长,使用斯坦福大学的在线引物设计软件对PVT1基因和NEAT1进行设计,以GAPDH作为对照;后进行反转录,通过实时定量聚合酶链反应(Real-time PCR)法检测食管鳞状细胞癌患者肿瘤组织和正常组织中PVT1和NEAT1的表达含量,结合患者临床资料,以spss19.0分析其表达含量与患者临床病理特征之间的关系。2搜集并提取TCGA数据库关于ESCC相关测序资料,利用oncolnc分析此两种LncRNA表达水平与患者生存之间的关系。结果1长链非编码RNA PVT1和NEAT1在食管鳞状细胞癌肿瘤组织中的表达高于正常组织,其表达含量的差异具有统计学意义(P0.05),2 LncRNA PVT1和NEAT1的表达水平的高低与患者年龄、性别、肿瘤部位等无明显相关性(P0.05),而与ESCC的淋巴结是否转移、肿瘤分化程度、TNM分期等明显相关(P0.05)3 Kaplan-Meier生存分析显示:与低表达组相比,PVT1和NEAT1高表达组的5年生存率下降(P0.05)。结论1长链非编码RNA PVT1和NEAT1在食管鳞状细胞癌肿瘤组织中的表达要明显高于正常组织。2高表达LncRNA PVT1和NEAT1的ESCC往往分期较晚,预后不良。3 LncRNA PVT1和NEAT1与食管鳞状细胞癌的发生发展密切相关,有可能作为新的分子标志物为食管癌的早期诊断和治疗服务。
[Abstract]:Background and objective esophageal cancer is a common malignant tumor. Its incidence and mortality rate are sixth and eighth respectively in China. The incidence of esophageal cancer is often caused by bad eating habits. It has two types of pathological types: esophageal squamous cell carcinoma and esophageal adenocarcinoma. In the case of discomfort symptoms such as dysphagia, the patients are found at the time of gastroscopy, while some of the patients are in the late stage. The main treatment for esophageal cancer is still the operation. The operation not only brings pain to the patients, but also may produce postoperative complications such as anastomotic fistula. Therefore, the early detection of esophageal cancer and the use of molecular mechanisms can be used for drug treatment. Cancer of the esophagus is an urgent need. Long chain noncoding RNA is a class of RNA sequences with a length of about 200 nucleotide lengths. Although it does not encode protein, it plays an important regulatory role in embryonic development, cell proliferation, differentiation and regulation of gene expression. Therefore, it is presumed that it is in the process of cancer development and development. It also plays an important regulatory role.PVT1 is one of the primary cancer LncRNA, about 300bp long and only 57kb from the MYC gene we know. Due to its special location, it is presumed that the two may play a coordinated role in regulating the occurrence and development of the tumor..NEAT1 is a long chain non coded RNA, located on the human chromosome first, and the immune response, gene expression. Regulation and other biological processes are related. Previous studies have confirmed that NEAT1 is up regulated in many kinds of tumors, such as glioma, leukemia, breast cancer, and larynx cancer. At present, the two LncRNA studies are mainly focused on other tumors, and the relationship with the cancer of the esophagus is rarely reported. Therefore, the aim of this paper is to detect long chain non coded RNA PVT1 and NE. The difference between the tumor tissue and normal tissue of the patients with esophageal squamous cell carcinoma (AT1), and the relationship between the expression content and the stage of esophageal cancer and the prognosis of the esophagus, are discussed as a new molecular marker for predicting the prognosis and treatment of esophageal cancer. Methods 1 long chain non coding RNA PVT1 and NEAT1 were used in the tumor tissue of the squamous cell carcinoma of the esophagus and the matched normal tissue. Differential expression study.1.1 collected 54 cases of esophageal squamous cell carcinoma in the Department of thoracic surgery of the Affiliated Hospital of Qiingdao University from June 2015 to April 2016. The normal tissues of the patients with esophageal squamous cell carcinoma (more than 5cm) and tumor tissues were collected. All patients were not treated with chemotherapy before operation. The pathological diagnosis of squamous cell carcinoma of the esophagus after operation, and the rapid resection of fresh cancer tissue quickly. In the EP tube containing RNA protection solution, and the cryopreservation.1.2 Trizol method, which was placed at -80 C in half an hour, to extract RNA in the tumor tissues and normal tissues of the squamous cell carcinoma of the esophagus respectively, the purity and concentration of the extracted RNA was detected by a micro spectrophotometer, and RNA between the suitable purity (1.80-2.0) was used as the next experiment. To meet the requirements of RE Extraction until it meets the requirements. The reverse transcription of LncRNA PVT1 and LncRNA NEAT1, downstream design primers, and the gene database in NCBI website are used to find the sequence of LNC RNA PVT1 and LNC RNA NEAT1, and the design software of the online primer design software of the Stanford University is used. As a control, the expression of PVT1 and NEAT1 in tumor tissues and normal tissues of patients with esophageal squamous cell carcinoma was detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the relationship between the expression content and the clinicopathological features of the patients was analyzed by spss19.0 to collect and extract TCGA by spss19.0. The database about ESCC related sequencing data and oncolnc analysis of the relationship between the two LncRNA expressions and the survival of the patients. Results the expression of 1 long chain non coded RNA PVT1 and NEAT1 in the tumor tissues of the squamous cell carcinoma of the esophagus was higher than that of the normal tissue, and the difference of its expression content was statistically significant (P0.05), 2 LncRNA PVT1 and NEAT1. There was no significant correlation between the level of expression and the age, sex, and tumor location of the patients (P0.05), but a significant correlation with the metastasis of ESCC lymph nodes, the degree of tumor differentiation, and the TNM staging (P0.05) 3 Kaplan-Meier survival analysis showed that the 5 year survival rate of the high expression group of PVT1 and NEAT1 decreased (P0.05). Conclusion the 1 long chain non coding group was compared with the low expression group. The expression of RNA PVT1 and NEAT1 in the tumor tissues of squamous cell carcinoma of the esophagus is obviously higher than that of normal tissue.2 high expression LncRNA PVT1 and NEAT1 ESCC often staging later. The poor prognosis.3 LncRNA PVT1 and NEAT1 are closely related to the development and development of squamous cell carcinoma of the esophagus. It may be a new molecular marker for the early diagnosis of esophageal cancer. And treatment services.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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