Spatial and single-cell transcriptomics reveal the regional division of the spatial structure of NASH fibrosis

Abstract Objective : To reveal the regional division of the spatial structure of NASH fibrosis and the communication relationship between cells in different regions and to analyse specific marker genes as potential therapeutic targets for NASH fibrosis. Methods: The liver sections of healthy controls, NAFL patients and NASH patients were measured by spatial transcriptomics, and integration analysis was performed with single-cell RNA-seq. Differential expression, functional pathway prediction, and deconvolution analysis revealed lineage-specific changes in gene expression, subpopulation composition and intercellular communication in NASH and identified key genes involved in HSC activation. The role of key genes in NASH fibrosis was verified in vitro and in vivo. Result : Descending and clustering analysis of 4114 nuclei from liver tissues of healthy controls, NAFL, and NASH showed that the distribution of cluster5 (fibrotic region) is dominated by lobules, and a small amount of fibrosis can be seen in the sink area. Functional analysis suggested that differentially expressed genes of cluster5 were concentrated in ECM structural components and signalling molecules. Six cell types were obtained by integrating the single-cell sequencing dataset (GSE189175). Compared with the healthy control and NAFL groups, the NASH group had significantly increased proportions of HSCs and myofibroblasts, which were distributed in the lobule and the portal area around the fibrotic area. Simultaneously, the infiltration of Kupffer cells around the fibrotic area also increased. The cell communication analysis showed that diffusive cell communication was the main type, including endocrine, paracrine and autocrine communication, followed by ECM-receptor cell communication. According to the analysis of differentially expressed genes in the subsets, AEBP1 and DPT are relatively highly expressed in cluster5, as well as in HSCs and myofibroblasts. SCENIC analysis found that AEBP1+ and DPT+ myoblasts were involved in the activation of HSCs and fibrosis formation. Immunohistochemistry verified the high expression of AEBP1 and DPT in patients with NASH fibrosis. After transfection of AEBP1 and DPT interference fragments in LX2 cells in vitro, the mRNA level of Collagen Ⅰ in cells was significantly lower than that of the siRNA-NC group and blank control group. Conclusion: Our study is the first to reveal lineage-specific changes in gene expression, subpopulation composition and cell communication in NASH fibrosis, providing new directions for potential therapeutic targets for NASH fibrosis.