Analysis of TGFbeta-regulated microRNAs in hESC stemness and differentiation

描述（由申请人提供）：TGF 2信号通路的Smad 2/3分支是维持人胚胎干细胞（hESC）和人诱导多能细胞（hiPSC）多能性所必需的。尽管它的意义，多能性的分子电路，以及分化，仍然知之甚少，在这两个系统。最近的工作，包括我们自己的，已经表明，胚胎特异性microRNAs（miRNAs）通过调节Smad 2/3通路在调节早期脊椎动物发育和HESC的命运决定中起着至关重要的作用。该通路作为形态发生素，转导激活素/结信号并基于信号传导的浓度和持续时间引发不同的结果。虽然在解决miRNA生物化学和作用机制的分子机制方面取得了巨大进展，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。这项赠款的两个具体目标是在这两个领域之间架起桥梁，以便继续努力对两者进行更全面的了解。本文提出的初步实验表明，不同水平的抑制正在进行的Smad 2/3信号（模仿形态发生作用），具体调节几个miRNA在hESC中的表达。基于这些发现，我建议研究这些Smad 2/3调控的miRNA在hESC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Smad 2/3信号的调节阈值所需的正确表达的每一个miRNA，其次是直接与间接之间的歧视，转录与转录后的调节机制。对每个阈值特异性miRNA的靶分析将解决它们的分子功能。其次，我们将直接在hESC和hiPSC中进行miRNA功能的获得和丧失。总之，本研究中提出的目标的成功实现将有助于更深入地了解从miRNA角度观察时阈值特异性Smad 2/3活性在hESC命运获取过程中的作用，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，鉴于癌细胞和胚胎干细胞之间的相似性以及TGF 2途径和miRNA在肿瘤发生中的既定作用，hESC和hiPSC中相关Smad 2/3调节的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.