Analysis of TGFbeta-regulated microRNAs in hESC stemness and differentiation

描述（由申请人提供）：TGF2信号通路的Smad2/3分支对于维持人胚胎干细胞（hESCs）和人诱导多能细胞（hiPSCs）的多能性是必需的。尽管具有重要意义，但在这两个系统中，多能性和分化背后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，已经表明胚胎特异性microRNAs （miRNAs）通过调节Smad2/3通路在调节早期脊椎动物发育和HESCs的命运决定中起着至关重要的作用。该通路作为形态因子，转导激活素/节点信号，并根据信号的浓度和持续时间引发不同的结果。虽然在miRNA生物化学的分子机制和作用机制的研究上取得了巨大的进展，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中的作用，仍然知之甚少。这笔赠款的两个具体目标是弥合这两个领域，以便我们继续努力更全面地了解这两个领域。本文提出的初步实验表明，对正在进行的Smad2/3信号传导的不同程度的抑制（模拟形态因子效应）特异性地调节了hESCs中几种mirna的表达。基于这些发现，我建议研究这些smad2 /3调控的mirna在hESCs和hiPSCs的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信号的调节阈值，然后区分直接与间接以及转录与转录后的调节机制。每个阈值特异性miRNA的目标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功能的获得和丧失。综上所述，本研究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，并建立这些选择性表达的miRNA在相同背景下的生物学功能。此外，考虑到癌细胞与胚胎干细胞的相似性，以及TGF2通路和mirna在肿瘤发生中的作用，在hESCs和hiPSCs中相关smad2 /3调控mirna的发现和功能表征将对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. PUBLIC HEALTH RELEVANCE: In this study we propose to investigate the regulatory mechanisms and the function of these Smad2/3-regulated miRNAs during both pluripotency and differentiation of hESCs. By targeting and resolving the role of the Smad 2/3 branch of the TGF2 pathway and their regulation of miRNAs in the context of hESC differentiation, these studies contribute to our basic understanding of molecular processes involved in fate determination in hESCs. Moreover, as this pathway is also involved in cancer, the resolution of the aims of this study will also contribute to our basic understanding of the molecular basis of tumor formation. Thus, the results obtained from the successful accomplishment of the aims of this grant will have a significant impact on biomedical science and public health.