Analysis of TGFbeta-regulated microRNAs in hESC stemness and differentiation

描述(申请人提供)：TGF2信号通路的Smad2/3分支对于维持人类胚胎干细胞(HESCs)和人类诱导多能细胞(HiPSCs)的多能性是必要的。尽管其意义重大，但在这两个系统中，潜在的多能性以及分化的分子电路仍然知之甚少。最近的工作，包括我们自己的工作，表明胚胎特异的microRNAs(MiRNAs)通过调节Smad2/3途径在脊椎动物早期发育和hESCs的命运决定中发挥关键作用。这条通路作为形态原，传递激活素/结节信号，并根据信号的浓度和持续时间而产生不同的结果。虽然在解决miRNA生物化学的分子机制和作用机制方面已经取得了巨大的进展，但它们在生物过程中的作用，特别是在早期胚胎发育和细胞命运决定过程中的作用，仍然知之甚少。这笔赠款的两个具体目标是连接这两个领域，以便继续努力更全面地了解这两个领域。本文提供的初步实验表明，对正在进行的Smad2/3信号的不同程度的抑制(模仿形态效应)特异性地调节了hESCs中几个miRNAs的表达。基于这些发现，我建议研究这些Smad2/3调控的miRNAs在hESCs和hiPSCs多能性和分化过程中的调控机制和功能。我们的第一个目标是确定每个miRNA正确表达所需的Smad2/3信号的调控阈值，然后区分直接和间接以及转录和转录后的调控机制。针对每个阈值特定miRNA的靶向分析将解决它们的分子功能。其次，我们将直接在hESCs和hiPSCs中进行miRNA功能的得失。综上所述，本研究提出的目标的成功实现将有助于从miRNA的角度更深入地了解阈值特异性Smad2/3活性在hESC命运获得过程中的作用，并在相同的背景下建立这些选择性表达的miRNA的生物学功能。此外，鉴于癌细胞和胚胎干细胞之间的相似性以及TGF2通路和miRNAs在肿瘤发生中的既定作用，hESCs和hiPSCs中Smad2/3调控的相关miRNAs的发现和功能鉴定将在癌症领域产生直接影响。

DESCRIPTION (provided by applicant): The Smad2/3 branch of the TGF2 signaling pathway is necessary to sustain pluripotency in human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs). Despite its significance, the molecular circuitry underlying pluripotency, as well as differentiation, remains poorly understood in both systems. Recent work, including our own, has shown that embryonic specific microRNAs (miRNAs) play crucial roles in modulating the fate decisions in early vertebrate development and HESCs by modulating the Smad2/3 pathway. This pathway acts as morphogen, transducing activin/nodal signals and eliciting different outcomes based on concentration and duration of signaling. While tremendous progress has been made in the resolution of the molecular mechanism underlying miRNA biochemistry and mechanism of action, their role in biological processes, specifically during early embryonic development and cell fate determination, also remains poorly understood. The two specific aims of this grant are to bridge these two areas in order to continue our efforts toward a more comprehensive understanding of both. Preliminary experiments presented here demonstrate that different levels of inhibition of ongoing Smad2/3 signaling (mimicking morphogen effects) specifically modulate expression of several miRNAs in hESCs. Based on these findings I propose to investigate the regulatory mechanisms and the function of these Smad2/3-regulated miRNAs during pluripotency and differentiation of hESCs and hiPSCs. Our first goal is to determine the regulatory threshold of Smad2/3 signaling required for the proper expression of each miRNA, followed by the discrimination between direct versus indirect, and transcriptional versus post-transcriptional mechanisms of regulation. Target analysis for each threshold-specific miRNA will address their molecular function. Secondly, we will perform gain and loss of miRNA function directly in hESCs and hiPSCs. Taken together, the successful accomplishment of the aims proposed in this study will contribute to a more in depth understanding of the role of threshold-specific Smad2/3 activity during hESC fate acquisition when observed from the miRNA perspective, and establish the biological function of these selectively expressed miRNA in the same context. Moreover, given the similarities between cancer cells and embryonic stem cells and the established role of both the TGF2 pathway and miRNAs in tumorigenesis, the discovery and the functional characterization of relevant Smad2/3-regulated miRNAs in hESCs and hiPSCs will have a direct impact in the cancer field.