长链非编码RNA LINC00638在食管鳞癌中的功能和作用机制研究

发布时间：2018-12-15 02:23

【摘要】：食管癌是我国常见的恶性肿瘤之一,在我国男性中的发病率排在第三位,女性中排在第五位。我国食管癌的病理类型多为食管鳞癌,目前对于食管鳞癌的治疗手段有效,病人预后差,五年生存率低。因此研究食管鳞癌发生发展的分子机制,为食管鳞癌的临床诊断和治疗提供分子标志物或药物靶点显得尤为重要。课题组前期通过对食管鳞癌组织样本进行全基因组测序和比较基因组杂交分析,发现了一个高频扩增区域14q32.2-32.33,且此区域的扩增与局部淋巴结转移密切相关。为了研究长链非编码RNA在食管鳞癌中的功能和作用机制,我们在此扩增区域筛选出一个在食管鳞癌细胞中表达,并且其表达与DNA扩增相关的长链非编码RNA,linc00638。通过RACE实验、Northern blot实验以及PCR实验,我们确定了 linc00638的全长核酸序列。在对食管鳞癌组织标本及其配对癌旁组织标本进行RNA原位杂交实验时发现,linc00638的细胞定位发生了变化:在166例癌旁组织中,linc00638定位于细胞核,而在84例癌组织中,linc00638定位于细胞核和细胞浆。Linc00638核浆均有分布的食管鳞癌组织样本对应着更高的淋巴结转移率。为了探究linc00638定位改变的分子机制,我们采用RNA pull-down实验联合质谱分析,发现PTB可以和linc00638结合,并调控linc00638的表达和细胞定位。体外的细胞实验表明linc00638可以促进食管鳞癌细胞的增殖、克隆形成、侵袭迁移,促进淋巴内皮细胞的迁移和成管。体内的动物实验进一步证实linc00638可以促进食管鳞癌细胞在动物体内的淋巴结转移。这些实验结果表明linc00638具有促癌作用。分子机制研究发现,位于细胞浆的linc00638可以直接与VEGF-C mRNA结合,并增加mRNA的稳定性,促进VEGF-C的表达;此外,linc00638还可以与miR-216b相互作用,调控miR-216b靶基因KRAS的表达。以上研究表明长链非编码RNAlinc00638在食管鳞癌的发生发展过程中发挥着重要的促进作用,为将来的食管鳞癌的诊断和治疗提供了理论基础和分子标志物。
[Abstract]:Esophageal cancer is one of the most common malignant tumors in China. Esophageal squamous cell carcinoma is the pathological type of esophageal cancer in China. At present, the treatment of esophageal squamous cell carcinoma is effective, the prognosis of patients is poor, and the 5-year survival rate is low. Therefore, it is very important to study the molecular mechanism of esophageal squamous cell carcinoma, to provide molecular markers or drug targets for the clinical diagnosis and treatment of esophageal squamous cell carcinoma. A high frequency amplification region of 14q32.2-32.33 was found by genomic sequencing and comparative genomic hybridization analysis of esophageal squamous cell carcinoma tissue samples, and the amplification of this region was closely related to local lymph node metastasis. In order to study the function and mechanism of long chain noncoding RNA in esophageal squamous cell carcinoma, we screened out a long strand noncoding RNA,linc00638. expressed in esophageal squamous cell carcinoma cells in this amplification region, and its expression was associated with DNA amplification. The full length nucleic acid sequence of linc00638 was determined by, Northern blot and PCR experiments in RACE experiment. RNA in situ hybridization of esophageal squamous cell carcinoma and its matched adjacent tissues showed that the cellular localization of linc00638 had changed: in 166 paracancerous tissues, linc00638 was located in the nucleus, while in 84 cancer tissues. Linc00638 was located in the nucleus and cytoplasm of the esophageal squamous cell carcinoma (ESCC) with the distribution of Linc00638 nucleus and cytoplasm. The higher lymph node metastasis rate was found in the samples of esophageal squamous cell carcinoma (ESCC). In order to explore the molecular mechanism of linc00638 localization, we found that PTB can bind to linc00638 and regulate the expression of linc00638 and cellular localization by RNA pull-down experiment combined with mass spectrometry. Cell experiments in vitro showed that linc00638 could promote the proliferation, clone formation, invasion and migration of esophageal squamous carcinoma cells, and promote the migration of lymphatic endothelial cells and the formation of tubules. Animal experiments in vivo further confirmed that linc00638 can promote lymph node metastasis of esophageal squamous cell carcinoma cells in vivo. These results suggest that linc00638 can promote cancer. The molecular mechanism showed that linc00638 located in cytoplasm could bind directly to VEGF-C mRNA, increase the stability of mRNA and promote the expression of VEGF-C. In addition, linc00638 could interact with miR-216b to regulate the expression of miR-216b target gene KRAS. These studies suggest that long chain noncoding RNAlinc00638 plays an important role in the development of esophageal squamous cell carcinoma and provides a theoretical basis and molecular marker for the diagnosis and treatment of esophageal squamous cell carcinoma in the future.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

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