【作者】 刘梅；

【导师】 徐宁志； 程书钧； 高燕宁； 林东昕；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2009， 博士

【摘要】 最近的研究表明,miR-17-92基因簇在许多恶性肿瘤(包括B细胞淋巴瘤和肺癌)中存在高表达和扩增。本课题首先研究了miR-17-92基因簇在食管癌中的表达情况。我们发现在28例配对的食管癌标本中,有21例食管癌组织的miR-17-92基因簇的mRNA表达水平明显高于对照正常组织,占标本总数的75%;且在7个食管癌细胞系中均有表达。过表达miR-17-92基因簇能够明显促进细胞在体内、外的生长。其次,通过miRNA特异性实时定量PCR技术,我们在7个食管癌细胞系中检测了属于该基因簇的五个成熟miRNAs的表达情况,结果表明在食管癌细胞系中表达水平最高的是miR-92-1,其次依次为miR-20a、miR-17-sp、miR-19a和miR-18a。通过转染锁核酸(locked nucleic acid,LNA)修饰的反义寡核苷酸分别抑制内源性的属于该基因簇的五个miRNAs,发现抑制miR-19a后可明显抑制食管癌细胞的生长并诱导细胞发生凋亡,而转染miR-17-5p、miR-18a、miR-20a和miR-92-1相应的反义寡核苷酸却没有如此显著的抑制作用。利用Real Time-PCR芯片检测了370个与人类凋亡相关的基因,和转染对照反义寡核苷酸相比,转染miR-19a的反义寡核苷酸后,在mRNA水平上明显上调(2倍以上)的基因一共有19个,其中尤以肿瘤坏死因子TNF最为明显,升高了12倍以上。结合miRNA靶基因权威预测网站TargetScan和Sanger搜寻,发现TNF的3’UTR有miR-19a可能的结合位点,且通过报告基因实验,我们证明了TNF确实是miR-19a的一个新的直接靶基因。综上所述,本研究结果表明:miR-17-92基因簇在食管癌中呈现高表达,且miR-19a可能通过调控TNF等基因参与食管癌的发展过程。我们科室前期的研究结果表明EB1(end-binding protein)可通过激活β-catenin/T-cell factor(TCF)通路来促进细胞生长。本课题通过构建EB1基因的四环素诱导表达系统进一步研究其在调节细胞生长中的作用机制。利用EB1诱导表达系统,可有效上调EB1的蛋白水平。高表达EB1可明显促进细胞的生长并增强细胞在软琼脂中的克隆形成能力。此外,EB1还可以促进293-T-REx细胞在裸鼠体内形成肿瘤。EB1可激活β-catenin下游靶基因c-Myc的表达并上调其转录活性。我们还发现,诱导高表达EB1可以上调Bcl-2的表达,从而抑制顺铂诱导的细胞凋亡。EB1促进细胞生长和抑制细胞凋亡的作用都可以(至少部分地)被ANTCF所拮抗。此外,利用RNAi下调c-Myc可以抑制EB1诱导的Bcl-2的表达。总而言之,EB1作为一个癌基因通过β-catenin/TCF通路促进细胞生长和抑制细胞凋亡。更多还原

【Abstract】 Recent studies have demonstrated the overexpression and amplification of miR-17-92 cluster in many malignant human cancers,including B-cell lymphomas and lung cancers.The purpose of this study was to investigate the expression of miR-17-92 cluster in esophageal squamous cell carcinoma for the first time.We detected miR-17-92 cluster was overexpressed in 21 of 28(75%) esophageal cancer samples and all 7 esophageal cancer cell lines.miR-17-92 cluster could promote cell growth in vivo and in vitro.By miRNA-specific quantitative real-time reverse transcriptase-polymerase chain reaction(miR-qRT-PCR),the highest expression level of these five miRNAs is miR-92-1, followed by miR-20a,miR-17-5p,miR-19a and miR-18a.However,inhibition of miR-19a by antisense oligonucleotides(ONs) could induce apoptosis.In marked contrast, antisense ONs against miR-17-5p,miR-18a,miR-20a and miR-92-1 did not exhibit such significant inhibitory effects.Through Human Apoptosis RT Profiler PCR Array 384HT (370 genes),we found 19 genes was up-regulated more than 2-fold,when transfected with miR-19a antisense ONs compared with the control scramble ONs.TNF was 12-fold up-regulated.Combined with bioinformatics target prediction,miR-19a has a complementarity to the 3’UTR of TNF mRNA.We also identified TNF as a novel direct target of miR-19a by reporter assay.Taken together,we conclude that miR-17-92 cluster is overexpressed in esophageal squamous cancer and miR-19a may involve in this tumorigenesis. Previously we showed that End-binding protein 1(EB1) may promote cellular growth by activatingβ-catenin/T-cell factor(TCF) pathway.To further investigate the role of EB1 in regulating cellular growth,we established an EB1-inducible expression system.The protein level of EB1 was significantly up-regulated upon doxycycline induction.We found that EB1 promoted cellular growth and resulted in a significant increase in colony formation.In addition,EB1 could induce tumor formation in nude mice,activateβ-catenin-dependent gene expression and up-regulate the transcriptional activity of c-Myc.We also showed that EB1 in this manner inhibited apoptosis of 293-T-REx cells upon cisplatin and up-regulated expression of Bcl-2,whereas delta N-TCF4,an inhibitor ofβ-catenin/TCF pathway,could completely or partially abolish the effects of EB1 on the promotion of cell growth and the inhibition of apoptosis activity. Moreover,knockdown of c-Myc by RNAi could abrogate up-regulation of EB1-dependent induction of Bcl-2 expression.Overall,EB1 acts as a potential oncogene via activatingβ-catenin/TCF pathway to promote cellular growth and inhibit apoptosis.更多还原