CircRNA MYLK/lncRNA H19通过miR-29作为ceRNA调控膀胱癌EMT机制研究

Recent researches have indicated that non encoding RNA performs an important regulatory function, which is closely related to EMT and metastasis of tumors. However, the mechanisms underlying non encoding RNA remain largely unknown so far. Pandolfi proposed ceRNA hypothesis that exist competitive endogenous RNAs(ceRNA) in cell. These RNAs (mRNA, lncrna, circRNA, pseudogenes, etc.) molecules can competitively combine with miRNA through microRNA response element (MRE) to regulate each other expression level,which reveals the novel mechanism of RNA interaction. Our previous microarray experiment and the bioinformatics study found that H19 circRNA MYLK/lncRNA were over-expressed and co-expressed with several important target genes of mir-29, and share the same MRE of miR-29 in bladder cancer. luciferase reporter assay preliminarily confirmed that H19 CircRNA MYLK/lncRNA could combine with the mir-29. Therefore, it is speculated that MYLK/lncRNA H19 CircRNA might be used as ceRNA competitive binding miR-29 to regulate the expression of its target gene, and thus play an important role in the tumorigenesis and development of bladder cancer. The project intends to further verify the scientific hypothesis that CircRNA MYLK/lncRNA H19 and mir-29 target gene coud competitively bind to mir-29, which increase the related target genes expressiones to promote bladder cancer EMT and metastasis by q-RT PCR, Northern blot, FISH, RIP, RNA pull-down and gain of function and loss of function experiments, etc in the cell, animal and clinical specimens. The research start a regulatory function from the point of view of the CircRNA MYLK/lncRNA H19 as ceRNA, it reveals the novel mechanism to promote the invasion and metastasis of bladder cancer, and provides the original scientific basis for the diagnosis and therpy of bladder cancer.

新近研究表明非编码RNA与肿瘤的EMT及转移密切相关，然而迄今对其作用机制知之甚少。我们前期芯片及生物信息学分析发现CircRNA MYLK/lncRNA H19在膀胱癌中过表达且与miR-29的几个靶基因共表达，并都具有miR-29应答元件，荧光素酶报告基因实验初步证实CircRNA MYLK/H19能与miR-29结合。因此，推测CircRNA MYLK/H19可能作为竞争性内源RNA（ceRNA）竞争性结合miR-29调节其靶基因表达，进而促进膀胱癌EMT及转移。拟进一步应用qPCR、FISH、RIP、RNA pull down及功能获得和缺失等实验在细胞、动物及临床标本上证明CircRNA MYLK/H19与miR-29靶基因竞争性结合miR-29从而上调其相关基因的表达促进膀胱癌EMT及转移的科学假说。旨在揭示其促进膀胱癌侵袭转移的新颖机制，为膀胱癌诊断和治疗提供原创性科学依据。

项目的背景.新近研究表明以微RNA（miRNA）、长链非编码RNA（1ncRNA）、环形RNA（circRNA）为代表的非编码RNA（non-coding RNA, ncRNA）与肿瘤（膀胱癌）发生发展过程密切相关，有望成为肿瘤诊断治疗的新靶点，然而迄今对其作用机制知之甚少。我们前期芯片及生物信息学分析发现CircRNA MYLK/lncRNA H19在膀胱癌中过表达且与miR-29的几个靶基因共表达，并都具有miR-29 应答元件，荧光素酶报告基因实验初步证实CircRNA MYLK/H19能与miR-29结合。因此，推测CircRNA MYLK/H19可能作为竞争性内源RNA（ceRNA）竞争性结合miR-29调节其靶基因表达，进而促进膀胱癌EMT及转移。旨在揭示其促进膀胱癌侵袭转移的新颖机制，为其诊断和治疗提供原创性科学依据。.主要研究内容.本项目拟在前期的工作基础上进一步应用qRT-PCR、 Northern blot、FISH（荧光原位杂交）、RIP（RNA结合蛋白免疫共沉淀）、RNA pull down、Western blot、双荧光素酶报告基因及功能获得和缺失等实验在细胞、动物及临床标本上证明CircRNA MYLK/lncRNA H19与miR-29的靶基因竞争性结合miR-29从而上调其相关基因的表达促进膀胱癌EMT及转移这一科学假说。.重要结果、关键数据.1．证明了CircRNA MYLK/lncRNA H19在膀胱癌中过表达通过促进膀胱癌上皮型间质型转化，进而促进膀胱癌转移。.2． 阐明了CircRNA MYLK/lncRNA H19促进膀胱癌上皮型间质型转化的分子机制。CircRNA MYLK/lncRNA H19通过与miR-29竞争性地与其结合来调控miR-29靶基因表达，使其DNMT3B、ITGB1（β1-integrin）、VEGFA等靶基因表达上调，进而调节EMT相关的因子（TGF-β）和通路，最终促进膀胱癌细胞EMT和转移。为膀胱癌的诊断、预防和治疗提供新的思路和理论依据。.3. 在国际杂志上发表IF＞4.0的SCI论文3篇, 培养研究生3名。.科学意义.揭示CircRNA MYLK/lncRNA H19促进膀胱癌EMT及侵袭转移的新颖机制，旨为膀胱癌诊断和治疗提供原创性的科学依据。