Molecular mechanism behind the hematopoiesis-enhancing effect of SRT3025. Mus musculus

We used wild-type 129 mice to understand the mechanism of action behind SRT3025’s hematopoiesis-enhancing effect. Transcriptome analysis of cKit+ Sca1+ Lin- cells (KSL) cells discovered that a list of genes changed their expression levels significantly after SRT3025 administration in wild-type mice. Most notably, the cell cycle regulator p21 was down-regulated by 2.1 fold after SRT3025 administration. It is possible that the transcriptional suppression of p21 by SRT3025 might contribute to the compound’s beneficial effects on hematopoiesis. It has to be pointed out that, since our transcriptome analysis was limited to hematopoietic stem and progenitor cell population, we cannot rule out the possibility that SRT3025 works through the regulation of other cells such as certain important HSC niche components. The HSC niche is known to regulate stem cell pool size. Among the other genes suppressed by SRT3025, Thbs1 and Fosl2 encode thrombospondin 1 and Fos-like antigen 2, respectively. Both proteins are components of the HSC niche. Overall design: The goal of this study is to investigate gene expression changes in wild-type 129 mice in response to SRT3025 treatment. The study focuses on bone marrow cKit+ Sca1+ Lin- cells (representing hematopoietic stem and progenitor cells). These cells were sorted twice by FACS to ensure the purity. Cells of interest were collected in Trizol. RNA were isolated using RNAeasy mini prep kit and mRNAs were positively selected using oligo(dT)- Dynobeads. Then RNAseq libraries were then made using Illumina TruSeq RNA Sample Prep Kit and sequeced on an Illumina HiSeq 2000 genome analyzer.

本研究使用野生型129小鼠，以解析SRT3025促进造血作用的分子机制。对cKit+ Sca1+ Lin-细胞（即KSL细胞）进行转录组分析后发现，野生型小鼠经SRT3025给药后，存在一批基因的表达水平发生显著变化。尤为值得注意的是，细胞周期调控因子p21在SRT3025给药后表达下调2.1倍。推测SRT3025对p21的转录抑制作用，可能是该化合物发挥造血获益效应的潜在机制之一。需说明的是，由于本次转录组分析仅局限于造血干祖细胞群体，我们无法排除SRT3025通过调控其他细胞类型发挥作用的可能性，例如部分关键的造血干细胞龛（HSC niche）组分相关细胞。已知造血干细胞龛可调控干细胞池的大小。在SRT3025下调的其他基因中，Thbs1与Fosl2分别编码血小板反应蛋白1（thrombospondin 1）和Fos样抗原2（Fos-like antigen 2），二者均为造血干细胞龛的组成成分。实验设计：本研究旨在探究野生型129小鼠经SRT3025处理后的基因表达变化，实验聚焦于骨髓来源的cKit+ Sca1+ Lin-细胞（代表造血干祖细胞）。该细胞群体通过流式细胞术（FACS）分选两次以确保纯度。目的细胞收集于Trizol试剂中，采用RNAeasy迷你提取试剂盒分离总RNA，并通过oligo(dT)-Dynobeads正向富集mRNA。随后使用Illumina TruSeq RNA样本制备试剂盒构建RNA测序文库，并在Illumina HiSeq 2000基因组分析仪上完成测序。