Data for the publication "An Integrative Transcriptome Subtraction Strategy to Identify Human lncRNAs That Specifically Play a Role in Activation of Human Hepatic Stellate Cells" https://doi.org/10.3390/ncrna10030034

Fibrotic liver features excessive deposition of extracellular matrix (ECM), primarily produced from “activated” hepatic stellate cells (HSCs). While targeting human HSCs (hHSCs) in fibrosis therapeutics shows promise, the overall understanding of hHSC activation remains limited, in part because it is very challenging to define the role of human long non-coding RNAs (lncRNAs) in hHSC activation. To address this challenge, we identified another cell type that acts via a diverse gene network to promote fibrogenesis. Then, we identified the lncRNAs that were differentially regulated in activated hHSCs and the other profibrotic cell. Next, we conducted concurrent analysis to identify those lncRNAs that were specifically involved in fibrogenesis. We tested and confirmed that transdifferentiation of vascular smooth muscle cells (VSMCs) represents such a process. By overlapping TGFβ-regulated lncRNAs in multiple sets of hHSCs and VSMCs, we identified a highly selected list of lncRNA candidates that could specifically play a role in hHSC activation. We experimentally characterized one human lncRNA, named CARMN, which was significantly regulated by TGFβ in all conditions above. CARMN knockdown significantly reduced the expression levels of a panel of marker genes for hHSC activation, as well as the levels of ECM deposition and hHSC migration. Conversely, gain of function of CARMN using CRISPR activation (CRISPR-a) yielded the completely opposite effects. Taken together, our work addresses a bottleneck in identifying human lncRNAs that specifically play a role in hHSC activation and provides a framework to effectively select human lncRNAs with significant pathophysiological role.

肝纤维化（Fibrotic liver）的特征为细胞外基质（extracellular matrix, ECM）过度沉积，而该基质主要由“活化”的肝星状细胞（hepatic stellate cells, HSCs）产生。尽管以人源肝星状细胞（human HSCs, hHSCs）为靶点的纤维化治疗策略已展现出应用前景，但学界对hHSCs活化过程的整体认知仍较为有限，部分原因在于阐明长链非编码RNA（long non-coding RNAs, lncRNAs）在hHSCs活化中的作用极具挑战性。为应对这一难题，我们首先鉴定出另一种通过多样化基因网络促进纤维化发生的细胞类型；随后，我们筛选出在活化hHSCs与上述促纤维化细胞中差异表达的lncRNAs。接着，我们通过联合分析鉴定出特异性参与纤维化进程的lncRNAs，并通过实验证实，血管平滑肌细胞（vascular smooth muscle cells, VSMCs）的转分化（transdifferentiation）正是符合该特征的过程之一。通过在多组hHSCs与VSMCs中重叠比对转化生长因子β（TGFβ）调控的lncRNAs，我们筛选得到一组高度精选的lncRNA候选分子，其可能在hHSCs活化中发挥特异性作用。我们对其中一种命名为CARMN的人源lncRNA进行了实验表征，发现其在上述所有实验条件下均受TGFβ显著调控。敲低CARMN可显著降低hHSCs活化相关的一系列标志物基因的表达水平，同时减少ECM沉积与hHSCs迁移；反之，通过CRISPR激活（CRISPR activation, CRISPR-a）技术上调CARMN的表达，则会产生完全相反的生物学效应。综上，本研究解决了筛选特异性参与hHSCs活化的人源lncRNAs这一瓶颈问题，并为高效筛选具有重要病理生理功能的人源lncRNAs提供了研究框架。