Epigenetic roles in regulation of cholesterol metabolism and CVD risk

描述(由申请人提供)：几行实验证据表明，在不同类型的细胞中，细胞内胆固醇稳态的某些方面的破坏(例如 巨噬细胞，�细胞)可导致2型糖尿病(T2 DM)和动脉粥样硬化性血管疾病(ASCVD)之前的病理过程。我们最近对纯化的人类单核细胞的转录研究证实了这些发现，并特别确定了一个共表达的胆固醇代谢转录网络(CMTN)，其变化与T2 DM和冠状动脉钙化(CAC，一种亚临床ASCVD指标)显著相关。这个网络包括11个基因，涉及协同上调胆固醇吸收和合成，以及下调胆固醇外流--一个预计会增加细胞内胆固醇的分子图谱。为了将这些有趣的观察转化为对人类健康有意义的改善，我们的目标是全面描述人类单核细胞中这一基因网络的表观遗传调节因子，并研究该网络及其调节因子如何与单核细胞内胆固醇以及T2 DM和ASCVD的发生有关。我们的主要重点将是通过microRNAs(MiRNAs)对这个网络的表观遗传调控。已经通过体外和动物模型很好地证实了一种特定的miRNA(miR-33)与其共转录的宿主基因SREBP2在胆固醇稳态中发挥关键作用。我们对373个人类单核细胞样本的初步数据表明，miR-33a的细胞内水平与整个基因网络的表达有关，并与细胞供者中流行的T2 DM有关。我们还鉴定了其他几个与基因网络表达相关的有前途的miRNA候选基因。基于这些初步数据，并利用表型良好的动脉粥样硬化多种族研究(MESA)队列与现有基因组数据、1,264个单核细胞样本的DNA甲基化和转录数据以及373个单核细胞样本的miRNA测序数据，我们现在建议使用下一代测序来额外量化剩余891个单核细胞样本中的miRNAs，以实现以下特定目标：1)表征1,264个MESA单核细胞样本中miRNAs与CMTN之间的关系；2)在1,264名MESA参与者中建立miRNAs与T2 DM和CAC的关联；3)在MESA参与者中复制与最引人注目的证据相关的miRNA；4)使用体外培养的人单核细胞，验证CMTN改变和相关miRNAs的功能后果。结合遗传学、表观遗传学、转录和临床数据以及体外实验研究，可能为调节胆固醇代谢和T2 DM和ASCVD的易感性提供新的机制见解，并为T2 DM和ASCVD的预防和治疗提供新的策略。

DESCRIPTION (provided by applicant): Several lines of experimental evidence indicate that disruption of certain aspects of intra-cellular cholesterol homeostasis in various cell types (e.g. macrophage, �-cell) can lead to pathological processes preceding type 2 diabetes mellitus (T2DM) and atherosclerotic vascular disease (ASCVD). Our recent transcriptomic study of purified human monocytes corroborates these findings, and specifically identifies a co-expressed cholesterol metabolism transcriptional network (CMTN) whose alteration is significantly associated with T2DM and coronary artery calcification (CAC, a subclinical ASCVD measure). This network includes 11 genes involved in coordinated up-regulation of cholesterol uptake and synthesis, and down-regulation of cholesterol efflux - a molecular profile expected to increase intracellular cholesterol. To translate these intriguing observations into meaningful improvements in human health, our goal is to comprehensively characterize the epigenetic regulators of this network of genes in human monocytes, and to investigate how this network and its regulatory factors relate to intra-cellular cholesterol in the monocytes and to the development of T2DM and ASCVD. Our principle focus will be on epigenetic regulation of this network by microRNAs (miRNAs). It is already well established via in vitro and animal models that one specific miRNA (miR-33) plays a critical role in cholesterol homeostasis in concert with its co-transcribed host gene, SREBP2. Our pilot data from 373 human monocyte samples indicate that intra-cellular levels of miR-33a is associated with expression of the entire gene network of interest in this proposal and with prevalent T2DM in the cell donors. We also identified several other promising miRNA candidates associated with expression of the gene network. Based on these preliminary data, and taking advantage of the well-phenotyped Multi-Ethnic Study of Atherosclerosis (MESA) cohort with existing genomic data, DNA methylomic and transcriptomic data on 1,264 monocyte samples, and miRNA sequencing data in a subset of 373 monocyte samples, we now proposes to additionally quantify miRNAs in the remaining 891 monocyte samples using next generation sequencing to achieve the following specific aims: 1) To characterize the relationship between miRNAs and the CMTN in 1,264 MESA monocyte samples; 2) To establish the association of miRNAs with T2DM and CAC in the 1,264 MESA participants; 3) To replicate miRNA associations with the most compelling evidence in an independent set of 562 MESA participants; and 4) To validate the functional consequences of the CMTN alterations and associated-miRNAs, using ex-vivo cultured human monocytes. The integration of genetic, epigenetic, transcriptional, and clinical data along with the ex-vivo experimental studies may provide novel mechanistic insights concerning the regulation of cholesterol metabolism and susceptibility to T2DM and ASCVD and lead to new strategies for prevention and treatment of T2DM and ASCVD.