长链非编码RNA H19在食管癌发生发展中的功能及机制研究

发布时间：2018-08-18 18:13

【摘要】：研究背景食管癌是世界上常见的恶性肿瘤,已经成为全球死亡的主要原因之一,且发病有明显的地域和组织学类型上的差异。上皮来源的食管癌有两种主要病理类型:食管鳞状细胞癌和食管腺癌。在中国,食管鳞状细胞癌是食管癌的主要病理类型,大约占总数的90%以上。尽管食管癌诊断及治疗已有较大的进展,但是食管癌预后仍较差,总体5年生存率仍然处于较低的水平。其主要原因是缺乏有效的早期食管癌检测方法及进行合理的规范化治疗。因此,如何筛选出更多有效的肿瘤标志物及寻找食管癌治疗的潜在靶点已成为目前基础和临床研究的主要方向。人类基因组中只有不到2%的序列为蛋白质编码,其他序列虽然不编码蛋白质,但超过90%能被转录成RNA。这些不能为蛋白质编码的RNA分子统称为非编码RNA。非编码RNA参与了多种生物学过程,并在肿瘤发生发展中发挥重要作用。非编码RNA基于规模大致分为两大类:短链非编码RNA和长链非编码RNA。作为非编码RNA中的新角色,长链非编码RNA在各种疾病尤其是肿瘤中起重要调控作用,成为肿瘤研究的热点。长链非编码RNA在多种层面上(表观遗传水平、转录水平及转录后调水平)调控基因的表达。大量证据表明多种长链非编码RNA在多类肿瘤中存在异常表达,在肿瘤的发生、发展过程中发挥着促癌或抑癌作用。HOX转录RNA(HOTAIR)是第一个被发现在原发性与转移性乳腺癌中具有反式转录调控作用的长链非编RNA。近年来,越来越多的研究表明长链非编码RNA的异常表达与肿瘤的发生、发展及侵袭转移有密切关系。因此,探索肿瘤相关长链非编码RNA以及与蛋白编码基因之间的相互关系已经成为肿瘤生物研究领域的重要课题。长链非编码RNA H19作为印记基因的非编码转录本产物,其父源印记母系等位基因表达,不具编码蛋白能力。新近研究表明,H19位点缺失导致其在膀胱癌、乳腺癌、肝癌等多种恶性肿瘤中呈现较高丰度的表达,发现其具有原癌活性。但也有研究认为H19具有抑癌活性,可以抑制肿瘤的恶性增殖、侵袭转移和肿瘤新生血管的形成。更加引人关注的是H19有望成为临床早期诊断和预后判断新的标志物,也为肿瘤的治疗提供了一种新的思路。目的长链非编码rnah19在食管癌中的生物学功能以及病理学机制尚不明确。本研究旨在探讨h19在食管癌发生、发展中的作用及其分子机制。材料方法1.收集本科室2007-2012年行肿瘤切除术的64例食管癌患者的组织标本,采用qrt-pcr的方法,定量检测了h19在64对食管癌组织及对应的正常食管组织中的表达水平,并分析h19的表达水平与患者临床病理参数的相关性。2.采用qrt-pcr的方法检测h19在正常食管上皮细胞株het-1a及食管癌细胞株ec109,ec9706,kyse150和kyse450中的表达水平,选择明显高表达的食管癌细胞株作为进一步研究的实验对象进行后续功能研究。3.通过sirna干扰技术沉默h19在食管癌细胞株ec109、ec9706中的表达水平,并通过cck-8法、细胞周期实验、裸鼠原位成瘤实验、transwell细胞迁移侵袭实验检测h19表达干扰后对食管癌细胞株的生长、迁移、侵袭等肿瘤细胞学行为的影响。4.经westernblot法分析h19沉默对emt上皮标志物e-cadherin,间质标志物vimentin及其mmps家族中mmp-9蛋白表达的影响。结果1.长链非编码rnah19在食管癌组织的表达水平均明显高于癌旁正常组织;在食管癌的发生、发展中可能担当重要的角色。其表达与患者肿瘤的浸润深度、淋巴结转移显著相关,与年龄、性别与肿瘤分化程度无关。2.本研究通过对4种食管癌细胞株及正常上皮细胞株中h19的检测,证实h19在食管癌细胞株里的表达水平也明显高于食管正常上皮细胞株。食管癌细胞株ec109及ec9706明显高表达h19,可以作为进一步研究的实验对象。3.通过sirna干扰技术沉默h19在食管癌细胞株ec109、ec9706中的表达水平后,cck-8法检测h19表达下调后可显著抑制食管癌细胞的增殖能力。流式细胞仪检测h19表达干扰后食管癌细胞株发生g0/g1期阻滞。为了进一步探讨h19在体内的促癌生长作用,我们利用h19干扰ec9706细胞株及阴性对照组进行了裸鼠皮下成瘤实验,发现h19干扰细胞株在裸鼠皮下的成瘤能力明显受到抑制,其肿瘤体积及重量均明显低于阴性对照组。我们进一步懫用transwell小室研究了h19表达干扰细胞株ec109和ec9706的迁移和侵袭能力,结果表明,h19表达干扰细胞株的迁移和侵袭能力较对照组明显抑制。4.Western blot结果显示,与阴性对照组比较,抑制H19表达后,食管癌细胞上皮细胞标志物E-cadherin表达水平明显增高,间质细胞标志物vimentin及MMP-9蛋白的表达水平下调。结论综上所述,本研究显示长链非编码RNA H19在食管癌组织中表达上调,在食管癌中作为促癌基因促进肿瘤细胞的生长和转移。其分子机制可能通过介导EMT的发生促进食管癌侵袭和转移,抑制H19的表达能够阻滞EMT的发生。长链非编码RNA H19在食管癌转移过程中有重要的调控作用,有可能成为食管癌治疗的一个潜在分子靶点。
[Abstract]:Background Esophageal cancer is one of the most common malignant tumors in the world and has become one of the leading causes of death worldwide. There are two major pathological types of epithelial esophageal cancer: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Although the diagnosis and treatment of esophageal cancer have made great progress, the prognosis of esophageal cancer is still poor, and the overall 5-year survival rate is still at a low level. Effective tumor markers and potential targets for esophageal cancer therapy have become the main directions of basic and clinical research. Only less than 2% of human genome sequences are encoded by proteins. Although other sequences do not encode proteins, more than 90% of them can be transcribed into RNA. Coded RNA. Non-coding RNA participates in many biological processes and plays an important role in tumorigenesis and development. Long chain non coding RNA regulates gene expression at various levels (epigenetic level, transcriptional level and post transcriptional regulation level). A large number of evidence shows that a variety of long-chain non coding RNA have abnormal expression in many kinds of tumors, and play a role in promoting cancer or inhibiting cancer in the process of tumor occurrence and development..HOX transcription RNA (HOTAIR) is the key point of tumor research. In recent years, more and more studies have shown that the abnormal expression of long-stranded non-coding RNA is closely related to the occurrence, development, invasion and metastasis of tumors. Therefore, to explore long-stranded non-coding RNA and protein-coding genes related to tumors. Long-stranded noncoding RNA H19, as a noncoding transcript product of imprinted genes, has no ability to encode proteins because of its paternally imprinted maternal allele expression. H19 has been shown to be highly expressed and has proto-oncogenic activity. However, some studies have suggested that H19 can inhibit the malignant proliferation, invasion, metastasis and neovascularization of tumors. Objective The biological function and pathological mechanism of long-chain non-coding rnah19 in esophageal cancer are still unclear. This study was designed to explore the role of H19 in the occurrence, development and molecular mechanism of esophageal cancer. Materials and Methods 1. The tissue specimens of 64 patients with esophageal cancer who underwent tumor resection in our department from 2007 to 2012 were collected and QR was used. T-pcr was used to quantitatively detect the expression of H19 in 64 esophageal cancer tissues and corresponding normal esophageal tissues, and to analyze the correlation between the expression of H19 and the clinicopathological parameters of patients. 2. the expression of H19 in normal esophageal epithelial cell lines het-1a and esophageal cancer cell lines ec109, ec9706, kyse150 and KYSE450 was detected by qrt-pcr. 3. Silencing the expression of H19 in esophageal cancer cell lines Ec109 and EC9706 by siRNA interference technique, and through CCK-8 method, cell cycle test, in situ tumorigenesis test in nude mice, Transwell cell migration and invasion test The effects of H19 silencing on the expression of MMP-9 protein in EMT epithelial marker e-cadherin, interstitial marker vimentin and its MMPs family were analyzed by Western blot. results 1. The expression of H19 may play an important role in the occurrence and development of esophageal cancer. The expression of H19 is significantly related to the depth of tumor invasion and lymph node metastasis, and is not related to age, sex and tumor differentiation. 2. The detection of H19 in four esophageal cancer cell lines and normal epithelial cell lines confirmed that H19 is present. Esophageal cancer cell lines Ec109 and EC9706 were significantly higher than normal esophageal epithelial cell lines. The proliferation of esophageal cancer cells was significantly inhibited. The g0/g1 phase arrest of esophageal cancer cell lines was detected by flow cytometry. In order to further explore the effect of H19 on the growth of esophageal cancer cells in vivo, we used H19 to interfere with EC9706 cell line and negative control group to carry out subcutaneous tumorigenesis test in nude mice. Subcutaneous tumorigenesis was significantly inhibited, and the tumor volume and weight were significantly lower than those in the negative control group. We further studied the migration and invasion of h19-interfering cell lines Ec109 and EC9706 by Transwell chamber. The results showed that h19-interfering cell lines migration and invasion were significantly inhibited. The results of blot showed that after inhibiting the expression of H19, the expression of E-cadherin, the epithelial markers, and the expression of vimentin and MMP-9 were down-regulated in esophageal cancer cells. As an oncogene promoting tumor cell growth and metastasis, long-stranded non-coding RNA H19 may play an important role in the regulation of metastasis of esophageal cancer and may be a potential therapeutic factor for esophageal cancer. Subtarget.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1

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