Single-Cell Transcriptome Analysis Identified Core Genes and Transcription Factors in Mesenchymal Cell Differentiation during Liver Cirrhosis.

BACKGROUND: Mesenchymal cells, including hepatic stellate cells (HSCs), fibroblasts (FBs), myofibroblasts (MFBs), and vascular smooth muscle cells (VSMCs), are the main cells that affect liver fibrosis and play crucial roles in maintaining tissue homeostasis. The dynamic evolution of mesenchymal cells is very important but remains to be explored for researching the reversible mechanism of hepatic fibrosis and its evolution mechanism of hepatic fibrosis to cirrhosis. METHODS: Here, we analysed the transcriptomes of more than 50,000 human single cells from three cirrhotic and three healthy liver tissue samples and the mouse hepatic mesenchymal cells of two healthy and two fibrotic livers to reconstruct the evolutionary trajectory of hepatic mesenchymal cells from a healthy to a cirrhotic state, and a subsequent integrative analysis of bulk RNA sequencing (RNA-seq) data of HSCs from quiescent to active (using transforming growth factor ß1 (TGF-ß1) to stimulate LX-2) to inactive states. RESULTS: We identified core genes and transcription factors (TFs) involved in mesenchymal cell differentiation. In healthy human and mouse livers, the expression of NR1H4 and members of the ZEB families (ZEB1 and ZEB2) changed significantly with the differentiation of FB into HSC and VSMC. In cirrhotic human livers, VSMCs transformed into HSCs with downregulation of MYH11, ACTA2, and JUNB and upregulation of PDGFRB, RGS5, IGFBP5, CD36, A2M, SOX5, and MEF2C. Following HSCs differentiation into MFBs with the upregulation of COL1A1, TIMP1, and NR1H4, a small number of MFBs reverted to inactivated HSCs (iHSCs). The differentiation trajectory of mouse hepatic mesenchymal cells was similar to that in humans; however, the evolution trajectory and proportion of cell subpopulations that reverted from MFBs to iHSCs suggest that the mouse model may not accurately reflect disease progression and outcome in humans. CONCLUSIONS: Our analysis elucidates primary genes and TFs involved in mesenchymal cell differentiation during liver fibrosis using scRNA-seq data, and demonstrated the core genes and TFs in process of HSC activation to MFB and MFB reversal to iHSC using bulk RNA-seq data of human fibrosis induced by TGF-ß1. Furthermore, our findings suggest promising targets for the treatment of liver fibrosis and provide valuable insights into the molecular mechanisms underlying its onset and progression.

[Experimental investigation of roles of STAT4 and SOCS3 in differentiation and cytokine secretion of T helper cells by RNA interference].

OBJECTIVE: To investigate the roles of signal transducer and activator of transcription 4 (STAT4) and suppressor of cytokine signaling 3 (SOCS3) in differentiation and cytokine secretion of T helper (Th) cells by separate silencing. METHODS: Th0 cells were induced to differentiate to Th1 or Th2 cells by interleukin 12 (IL-12) or interleukin 4 (IL-4). The mRNAs of STAT4 and SOCS3 were silenced separately by small interfering RNA (siRNA) in Th0, Th1 and Th2 cells and then the lymphocytes cultured in Th1 or Th2 medium with IL-12 or IL-4 for another 48 h before cells and supernatants were collected for the detection of Th1 and Th2 type cytokines by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). By comparing different amounts of cytokines between before and after silencing the target mRNAs, the roles of STAT4 and SOCS3 in the differentiation and cytokine secretion in T helper cells were evaluated. RESULTS: Th1 type cytokine significantly decreased and Th2 type cytokine increased after silencing STAT4 in Th0 and Th1 cells; minimal changes were detected in both Th1 and Th2 type cytokines after silencing SOCS3. CONCLUSION: STAT4 mediates the IL-12-induced differentiation of T cells to Th1 subset and maintains the cytokine secretion of Th1 cells; STAT4 directly inhibits the development and cytokine secretion of Th2 cells; STAT4 deficiency results in impaired development and cytokines secretion of Th1 cells and the enhanced development and cytokines secretion of Th2 cells. SOCS3 has minimal effects on IL-12 or IL-4 induced differentiation and cytokine secretion of T helper cells.