Study of the effects of RNA splicing inhibitor Pladienolide B on hepatocyte proliferation in vitro

In this study, we explored the effects of RNA splicing on hepatocyte fate decision. We found spatial remodeling of hepatic lobule during liver regeneration by spatial transcriptome analysis in regenerative and control mouse model. We also reported cell heterogeneity of hepatocyte-organoids and found that splicing factors reprogram hepatocytes from single-cell resolution. To explore the detailed function of splicing factors, splicing inhibitors (Pladienolide B) were used to treated organoids, then total RNA were harvest for bulk RNA sequence. In summary, we found that splicing factors can promote the transformation of hepatocytes from metabolic state to proliferative state by spatial transcriptome, single-cell RNA sequencing, and bulk RNA sequencing. Overall design: To investigate function of RNA splicing in regulating hepatocytes fate determination, Pladienolide B and DMSO were used to treat hepatocyte-organoid. We then performed gene expression profiling analysis using data obtained from RNA-seq of 6 different samples containing 3 DMSO treated and 3 Pladienolide B treated.

本研究探究了RNA剪接（RNA splicing）对肝细胞命运决定的影响。通过对再生模型与对照小鼠开展空间转录组（spatial transcriptome）分析，本研究发现肝再生过程中肝小叶发生了空间重塑。此外，本研究还报道了肝细胞类器官的细胞异质性（cell heterogeneity），并证实剪接因子可在单细胞分辨率水平下对肝细胞进行重编程。为明确剪接因子的具体功能，本研究使用剪接抑制剂普拉迪诺利德B（Pladienolide B）处理肝细胞类器官，随后收集总RNA用于批量RNA测序（bulk RNA sequencing）。综上，本研究通过空间转录组、单细胞RNA测序（single-cell RNA sequencing）及批量RNA测序分析，证实剪接因子可促进肝细胞从代谢状态向增殖状态转化。
整体实验设计：为探究RNA剪接在肝细胞命运决定调控中的作用，本研究分别使用普拉迪诺利德B与二甲基亚砜（DMSO）处理肝细胞类器官。随后，针对6份样本的RNA测序（RNA-seq）数据开展基因表达谱分析，其中3份为二甲基亚砜处理组样本，剩余3份为普拉迪诺利德B处理组样本。