miR-8和Let-7a调控Wnt信号通路对涡虫再生的影响及机制研究

Stem cells hold great potential for regenerative medicine, but there's still a lot of problems about culture difficulties and high risks. In order to overcome these problems, it is pivotal to obtain a clearer understanding of the molecular mechanisms that underlie every aspect of stem cell biology in vivo. Because these studies are relatively limited in mammalian, planarians represent a suitable model or dissecting relevant molecular details. Freshwater planarian possesses extraordinary ability to regenerate lost body parts after amputation, pluripotent adult somatic stem cells, called neoblasts, assure this regenerative ability. Long lasting studies on planarian regeneration resulted in numerous observations concerning cellular level and molecular aspects. Indeed, the significance of Wnt signalling in the development was recently underscored by the discovery that β-catenin is responsible for regulating head vs. tail development and neuronal organisation during planarian regeneration, and that Wnts are involved throughout the initial wounding response. Wnt/β-catenin signaling pathway plays a pivotal role in the maintenance of pluripotency as well as in the process of somatic cell reprogramming. Intrinsic factors regulating pluripotency in stem cells include the transcription factors Oct4, Sox2, c-Myc and Nanog, ectopic expression of the these transcription factors can reprogram somatic fibroblasts to pluripotent iPS cells. .In previous studies, we researched differences expression of miRNA through miRNA microarray, deep sequence and small RNA library, and analyzed changes transcriptome profiling and digital gene expression of mRNA during different periods of regeneration. The results shows that miR-8 and Let-7a were significantly repressed in regeneration tissues, and they perhaps regulates regeneration through targeted β-catenin, Tcf and Fzd of Wnt pathways according to the miRNA-mRNA regulation analysis. Moreover, miR-8 and Let-7a also directly or through the Wnt to regulate SOX2, Oct4, lin28, c-MYC and Nanog, transcription factors of required for stem cells self-renewal. This strongly suggested that let-7a and miR-8 were importment regulator for planarian regeneration, stem cells self-renewal and differentiation..Based on the previous results of miRNA-mRNA regulation analysis, the project to research expression profile (phases, tussues) and function (regulation mechanism) analysis of Let-7 and miR-8 and its target genes at planarian regeneration and stem cells pluripotency, and to examin the possibility of somatic cell reprogramming in vivo through lose-of-function of miRNAs(miR-8, Let-7a) and/or overexpression of transcription factors in planarian.In this way the fruits of the research will be helped to understanding the molecular principles governing the microRNA-mediated regulatory mechanisms of planarians regeneration, provides new ideas and useful information to the field of regenerative medicine base on stem cells.

涡虫作为理想的再生和干细胞研究模式生物，具有大量成体干细胞和强大的再生能力，Wnt信号是其再生的重要调控通路，但分子机制不清。我们研究发现miR-8和Let-7a可调控涡虫Wnt通路；文献报道SOX2、Oct4、lin28和c-MYC等受Wnt调控，也是miR-8或Let-7a的靶。我们推测miR-8、Let-7a通过直接或间接（Wnt通路）调控上述转录因子而影响涡虫再生和干细胞特性。本研究拟通过定量PCR、Northern、原位杂交等方法研究miR-8、Let-7a及靶在再生各时期、部位的表达特征，验证调控关系；通过miRNA mimics、miRNA antagomirs和RNAi研究miR-8、Let-7a对Wnt通路、再生和干细胞的调控机制及靶基因功能；探讨通过对miRNA和转录因子的表达控制在活体水平进行涡虫体细胞重编程的可能性，为干细胞和再生医学研究提供新的研究思路和有用信息。

涡虫作为理想的再生和干细胞研究模式生物，具有大量成体干细胞和强大的再生能力， Wnt信号是其再生的重要调控通路。本项目中我们发现Let-7a 在再生过程中具有显著差异表达，并与 Wnt信号相关基因紧密关联；靶基因预测显示其与β-catenin 和 Fzd 的 mRNA 3’UTR 有 3 个结合位点； 细胞转染实验显示Let-7a 能够显著下调连有β-catenin 3’UTR 的 GFP（报告基因）荧光信号；向涡虫体内注射 Let-7a 模拟物（miRNAmimics）后，再生的涡虫产生两端均有眼点，形成与下调 Wnt 信号相类似的表型缺陷；以上结果表明，Let-7a通过负调控β-catenin 分子活性，影响 Wnt信号，调控干细胞的增殖、分化，进而调控涡虫的再生，圆满完成了申请时的计划。同时，还对与wnt信号通路具有crosstalk的TGF-β/Smads信号通路调控机制进行了初步研究，发现了TGF-β/Smads信号通路上Djsmad-4基因在再生过程中具有显著差异表达，敲低后可显著阻断涡虫再生，且神经系统再生发生异常；原位杂交结果显示Djsmad-4在再生过程中表达于干细胞富集的胚基处，提示TGF-β/Smads信号通路可能通过调控涡虫干细胞增殖、分化，影响神经系统的再生，最终导致再生异常，并利用差异转录组测序的方法，分析了TGF-β/Samds信号通路信号降低，对涡虫基因表达的影响，初步阐明了TGF-β/Smads信号通路调控涡虫干细胞增殖与分化，以及神经再生的分子机制。