Laboratory of Functional Biology (LFBio), Center for Cell-Based Therapy (CTC), Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, SP, CEP: 14051-140, Brazil.
Department of Genetics and Internal Medicine, Ribeirao Preto Medical School, University of São Paulo (FMRP-USP), Ribeirão Preto, SP, Brazil.
Stem Cell Res Ther. 2019 Jul 8;10(1):202. doi: 10.1186/s13287-019-1318-6.
By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated.
We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation.
Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states.
We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.
通过对多个靶标转录本进行转录后调控,microRNAs(miRNAs 或 miR)发挥着重要的生物学功能。H1 胚胎干细胞(hESCs)和 NTera-2 胚胎癌细胞(ECCs)是两种最广泛使用的人类多能性模型细胞系,它们具有一些共同的特征,包括与多能性状态或分化相关的 miRNA 的表达。然而,这些 miRNA 中的每一种如何在功能上影响这些细胞的生物学特性尚未被系统评估。
我们通过转染 miRNA 模拟物来研究 31 种 miRNA 对 NTera-2 和 H1 hESCs 的影响。培养 3-4 天后,用多能性标志物 OCT4 和 G2 细胞周期标志物 Cyclin B1 对细胞进行染色,并用 Hoechst 和 Cell Mask Blue 分别对细胞核和细胞质进行共染色。通过使用自动化定量荧光显微镜(即高通量筛选(HCS)),我们在两个细胞区室中获得了多个形态和标记强度测量值,生成了一个多参数 miR 诱导的表型谱,描述了与增殖、细胞周期、多能性和分化相关的变化。
尽管两种细胞类型总体上相似,但有些 miRNA 引起了细胞特异性的效应,而有些相关的 miRNA 则在同一细胞中引起了相反的效应。通过鉴定预测被诱导相似效应的 miRNA 共同靶向的转录本(通过层次聚类进行分组的 miRNA 谱),我们能够揭示潜在的调节信号通路和生物学过程,这些过程可能介导了 miRNA 对不同鉴定群组的影响。具体来说,我们表明 miR-363 通过靶向 NOTCH1 和 PSEN1 并抑制 Notch 诱导的分化来维持多能性,这一机制可能与原始和初始多能性状态有关。
我们提出了人类多能性细胞中首次进行的多参数高通量 miRNA 功能筛选。将这种类型的数据与使用相同 HCS 测定法从 siRNA 筛选中获得的类似数据进行整合,可以提供一种大规模的功能方法来识别和验证控制多能性和分化的 miRNA 介导的调控机制。