【作者】 丁大朋；

【导师】 赫捷；

【作者基本信息】 北京协和医学院 ， 肿瘤学， 2011， 博士

【摘要】 食管癌是一种常见的恶性肿瘤,在世界范围内的发病率和死亡率分别排在第8位和第6位。我国食管癌发病率居世界首位,近年来其发病率虽然有所下降,但死亡率变化不大,术后五年的生存率只有20-30%。食管癌的发生发展是一个多阶段,多种分子参与的复杂过程。然而,这些分子是如何调控食管癌的起始和进展,以及如何利用这些分子进行靶向治疗仍然需要进一步探索。miRNAs是长度为19-22 nt,高度保守的非编码RNA,并且能够通过转录后调控的机制抑制靶基因的翻译。miRNAs在细胞增殖,分化和凋亡过程中起到了重要的作用。最近越来越多的证据显示miRNAs在肿瘤的发生和发展中起到了重要的作用。我们前期芯片结果证明miR-29c在食管癌中明显的低表达。我们研究的口的是揭示miR-29c在食管癌中的表达模式及其在食管癌发生中发挥的作用。已有大量证据显示某些miRNAs的异常表达可能导致肿瘤的发生,并且在多种肿瘤中已经发现miR-29c低表达的现象。然而,miR-29c低表达在食管癌中所发挥的作用仍然不清楚。在我们的研究中证明miR-29c在食管癌组织和细胞中明显低表达,并且能够在体内和体外抑制食管癌细胞的增殖。我们通过双荧光素酶报告基因实验证明cyclin E是miR-29c的靶基因。众所周知,cyclin E是细胞周期G1/S期过渡的重要分子,并且其在食管癌的发生过程中频繁的高表达。实验结果证明在食管癌细胞中miR-29c和cyclin E的表达水平呈负相关。同时,过表达miR-29c能够抑制cyclin E的表达,并且随着miR-29c前体分子转染剂量的增加而cyclin E的表达降低。此外,我们还发现miR-29c过表达能够诱导细胞周期G1/S期阻滞并且抑制Rb蛋白的磷酸化。这些结果暗示miR-29c可能是通过cyclin E/Cdk2-pRb信号通路实现的诱导细胞周期G1/S期阻滞。我们还检测其它的G1期相关蛋白的表达水平如cyclin D1、cyclin D2、CDK2、CDK6,结果证实miR-29c不影响这些蛋白的表达。因此,我们可以得出miR-29c在食管癌细胞中主要通过调控cyclin E的表达诱导细胞周期G1/S期阻滞。综上所述,我们的研究证明miR-29c能够抑制cyclin E的表达,并且在食管癌的发生中起到抑癌分子的作用。近期的文献报道,cyclin E在食管癌组织和细胞中持续高表达,我们的实验结果证明miR-29c通过作用在cyclin E 3’UTR抑制cyclin E的表达,这一机制为解释cyclin E在食管癌中持续高表达提供了新的线索。同时,miR-29c可能成为靶向治疗食管癌的一个重要分子,因此,miR-29c将具有良好的应用前景。更多还原

【Abstract】 Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer deaths worldwide. The incidence of esophageal squamous cell carcinoma (ESCC) is more frequent in China. Despite the advances in therapy; ESCC is still one of the most lethal malignancies in China, with an overall 5-year survival rate of 20-30% after curative surgery. The development of ESCC is a multistep, progressive process, and a number of canonical genetic alterations in the tumor cells have been identified. However, how specific molecules regulate the initiation and progression of esophageal squamous cell carcinoma and are utilized for a clinical target therapy remains to be elucidated.miRNAs are 19-22 nt in length, highly conserved noncoding RNA that control gene expression post-transcriptionally. miRNAs play important roles in cell proliferation, differentiation, and apoptosis. Recent cumulative evidences suggested that miRNAs play important roles in the tumor growth and development. Our previously microarray analysis demonstrated that miR-29c expression was down-regulated in esophageal squamous cell carcinoma. The aim of this study was to evaluate the miR-29c expression patterns and the role of miR-29c in esophageal squamous cell carcinoma.Increasing evidence has suggested that dysregulation of certain miRNAs may contribute to tumorigenesis. Downregulation of miR-29c was observed in various types of cancers. However, the biological function of miR-29c in esophageal squamous cell carcinoma (ESCC) is still unknown. In our study, we showed that the expression of miR-29c was significantly decreased in ESCC tissues and five ESCC cell lines. Moreover, miR-29c could suppress in vitro and in vivo proliferation of ESCC cells. Cyclin E, which was critical for G1/S transition and was overexpression in most of esophageal squamous cell carcinoma, was identified as a target of miR-29c by luciferase assay. The cyclin E protein expression was detected in ESCC cell lines by western blotting. Interestingly, inverse correlations between miR-29c and cyclin E protein level were found in five ESCC cell lines. Introduction of miR-29c could reduce the expression of cyclin E protein but not the cyclin E mRNA, and the expression of cyclin E was decreased along with the increased miR-29c level. In addition, we observed that increased expression of miR-29c could induce cell cycle G0/G1 arrest and suppress the phosphorylation of Rb in ESCC cell lines. These results suggest that miR-29c may regulate G1/S transition through the cyclin E/Cdk2-pRb signaling pathway. We detect the expression of other G1 related protein such as cyclin Dl, cyclin D2, CDK2, and CDK6. The results confirmed that overexpression of miR-29c can not affect the expression of these proteins in ESCC cell lines. Furthermore, miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating expression of cyclin E mainly.Taken together, our study demonstrates that the miR-29c can regulate cyclin E and function as a tumor suppressor in ESCC. Recent evidences show the level of cyclin E protein has been found to increase in ESCC tissue and cell lines. miR-29c inhibits the translation of cyclin E mRNA by targeting its 3’UTR, which add a new explain of deregulation of cyclin E expression in ESCC. Therefore, miR-29c may serve as a useful therapeutic agent for miRNA-based ESCC therapy.更多还原