Decoding the Role of H19 in Cholestatic Liver Injury Using snRNA-seq, Spatial Transcriptomics, and Machine Learning-Based Disease Prediction.

Background: Primary Sclerosing Cholangitis (PSC) is a chronic obstructive biliary disease and remains a high-burden cholestatic liver disease with no approved therapies and a substantial recurrence rate following liver transplantation. The long non-coding RNA H19 (H19) has emerged as a potential driver of PSC progression, yet its cell-type-specific and spatially resolved mechanisms remain poorly defined. Results: Age- and sex-matched wild type (WT), H19 knockout (H19KO), Mdr2 knockout (Mdr2KO), and double-knockout (DKO; Mdr2KO/H19KO) mice were used. The liver tissues were analyzed using single nucleus RNA sequencing (snRNAseq) and NanoString GeoMx spatial transcriptomics to elucidate H19-dependent cellular and spatial alternations in cholestatic liver injury. Machine learning models (logistic regression, XGBoost, neural network, and random forest) were developed to generate cell-type specific disease prediction signatures and validated using the publicly available human dataset GSE243981. Both spatial transcriptomics and snRNAseq identified a disease-associated cholangiocyte subcluster that was significantly expanded in Mdr2KO mice, but markedly diminished in DKO mice, demonstrating a requirement for H19 in sustaining pathogenic cholangiocyte state. SPP1 signaling was significantly dysregulated in cholestatic liver injury and ameliorated with H19 deletion. Novel murine markers were identified, including Gm13775 (healthy hepatocytes) and Clu and Spp1 (healthy cholangiocytes), all of which were markedly downregulaed in disease. Machine learning-based, cell type-specific disease prediction models achieved AUC values > 0.87 when validated in the GSE243981 human dataset. Noteably,Spp1 expression decreased in cholangiocytes but was ectopically upregulated in hepatocytes in diseased liver, highlighting disrupted intercellular signaling network. Spatial analyses showed that Conclusion: H19 deletion mitigates cholestatic injury by suppressing pathogenic cholangiocyte states, normalizing SPP1-mediated signaling, and restoring bile-duct-localized transcriptional programs. These findings position H19 as a critical regulator of cholangiocyte-driven pathology and a potential therapeutic target in PSC.