miR-342/DNMT1/EVL(miR-342)反馈环调控大肠癌增殖、转移的机制研究

Our previous studies have shown that miR-342, a microRNA encoded in an intron of the gene EVL, inhibits DNMT1 expression by binding the 3 '-untranslated region of DNMT1 mRNA, then regulates the expression of certain tumor suppressor genes and metastasis related genes, thereby inhibits the proliferation and invasion of colorectal cancer cells. The expression of miR-342 is commonly suppressed in human colorectal cancer. That may result from the high methylation of CpG islands in upstream of EVL gene, the host gene of miR-342. In our preliminary experiments, it was found that the decrease of DNMT1 expression, by transfection of DNMT1 siRNA or miR-342, improved the transcription of EVL gene. So we speculate that there is a regulation system, miR-342/DNMT1/EVL(miR-342) feedback loop, in colorectal cancer. .In this project, the lentiviral inducible expression systems of miR-342, the cDNAs or shRNAs of DNMT1 and EVL will be utilized to investigate whether miR-342/ DNMT1/ EVL(miR-342) feedback loop exists in colorectal cancer cells or not, and seek the novel target genes directly regulated by miR-342 or DNMT1 by using the Illumina chip methylation assay, mRNA microarray, methylation specific PCR, methylation sequencing assay, quantitative RT-PCR, 2-D PAGE, etc. And clinical samples and data, combined with colorectal cell lines, will also be used to further clarify the effects and molecular mechanisms of the feedback loop in regulating epigenetic modification, cell proliferation and invasion of colorectal cancer.

我们前期研究表明miR-342能通过结合DNMT1 mRNA的3'非翻译区抑制其表达，藉此调节某些抑癌基因、肿瘤转移相关基因的表达，进而抑制大肠癌细胞增殖和侵袭。miR-342在大肠癌组织中低表达，这可能与其宿主基因EVL启动子CpG岛的高甲基化有关。我们的预实验发现通过转染siRNA或miR-342抑制DNMT1表达能提高EVL基因转录，推测大肠癌细胞中可能存在miR-342/DNMT1/EVL(miR-342)反馈调控环。本项目拟利用可诱导表达miR-342、DNMT1和EVL cDNA或shRNA的大肠癌细胞株和临床标本，通过芯片技术、甲基化特异PCR、甲基化测序、qPCR等方法，进一步证实miR-342/DNMT1/EVL(miR-342)反馈环的存在，发现miR-342和DNMT1调控的新的靶基因，阐明该反馈环在大肠癌表观遗传修饰、大肠癌增殖和侵袭中的作用及其分子机制。

研究背景：miR-342是由EVL基因内含子编码的，在大肠癌中普遍低表达，启动子高甲基化是miR-342低表达的原因之一。DNMT1是miR-342的直接靶基因，大肠癌中miR-342表达下调可能是DNMT1高表达的分子机制之一。miR-342能通过靶向抑制DNMT1表达，进而通过启动子去甲基化激活ADAM23、RECK及p21等抑癌基因表达，抑制大肠癌细胞的增殖和侵袭。.研究目的和方向：研究细胞中是否存在miR-342/DNMT1/miR-342反馈调控环及其在大肠癌增殖和转移中的作用；筛选miR-342直接靶基因并研究其在大肠癌中的作用机制；筛选miR-342通过DNMT1间接调控的靶基因，研究其对大肠癌增殖和转移的影响；在大肠癌临床标本研究上述调控作用的临床意义。为大肠癌发展和转移的表观遗传学研究提供理论基础。.研究结果：发现大肠癌中细胞存在miR-342/DNMT1/miR-342反馈调控环，其在大肠癌的发展和转移中发挥重要作用；FoxM1、DTNBP1、FBXO9、FoxQ1、ANLN、FMNL2、CCNF和ATP11A均是miR-342的直接靶基因；miR-342靶向抑制FoxQ1和ANLN表达，进而抑制大肠癌细胞的体外迁移和侵袭；miR-342通过靶向FoxQ1抑制大肠癌细胞发生上皮间质转化（EMT）；大肠癌组织和细胞中由于启动子甲基化导致miR-212表达下调，进而导致其靶基因MnSOD过表达，从而导致大肠癌细胞发生EMT和转移。临床标本研究发现miR-342、miR-212的低表达以及DNMT1的高表达与大肠癌转移及其不良预后相关。.结论：细胞中存在DNMT1/miR-342双负反馈调控环，大肠癌中miR-342下调导致其众多靶基因（癌基因）表达增高，而高表达的DNMT1通过调控启动子甲基化减少抑癌基因表达，这可能是大肠癌发生、发展的重要因素之一。