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WILMS TUMOR 1 PROMOTES THE BRIDGING FIBROSIS FORMATION THROUGH THE INVASIVE HEPATIC STELLATE CELL INDUCTION IN LIVER FIBROSIS
Methods: One-year-old Mdr2-/- mice (FVB background) and age-/gender- matched wild-type (WT) mice were used in this study. The serum exosomes were isolated using qEV isolation system (from IZON). Total RNA was isolated from the serum exosomes and liver tissues using Trizol. Total RNA transcriptome was determined by RNAseq. The miRNA profiles in the exosomes and livers were analyzed using the NanoString nCounter® miRNA Expression panel. TargetScan and mirnet2.0 were used to identify the potential target genes. Protein-protein interaction (PPI) was constructed based on the STRING database. Hub genes under cholestatic conditions were visualized by Cytoscape software. The human CCA patient data were downloaded from the Cancer Genome Atlas (TCGA) database.
Results: Bioinformatic analysis identified 74 differentially expressed miRNAs (DE-miRs) and 137 DE-miRs in the male and female Mdr2-/- mice compared to corresponding WT controls, respectively. The identified DE-miRs were used to predict the target genes. GO and KEGG analysis further showed that these DE-miRs were linked to multiple signaling pathways related to cholestatic liver injury. We also identified 431 up-, and 1106 down- DE genes (DEGs) in the Mdr2-/- mice compared to WT mice. From the PPI network analysis of the DEGs, the top 100 hug genes were determined. We identified 11 upregulated DE-miRs in Mdr2-/- mice, which overlapped with the predicted miRs based on the the top 100 hug genes. Interestingly, among the overlapping DE-miRs, two miRs, miR-16 and let-7e, were also upregulated in the serum exosomes of Mdr2-/- mice. Among the 100 hug genes, we identified nine downregulated genes that were potential targets of miR-16 and let-7e. Further analysis of TCGA CCA data showed upregulation of miR-16 and let-7e, and 7 out of the 9 identified targeted genes were also significantly downregulated in CCA compared to healthy controls.
Conclusion: This study identified potential DE-miRs in the liver and serum exosomes which may contribute to cholestatic liver injury. This study supports the possible use of exosomal miRs as diagnostic and prognostic markers and therapeutic targets for cholestatic liver diseases.

Fig 1. Liver DE-miRs and DEGs identification in Mdr2-/- mice compared to corresponding WT controls, target prediction and analysis of these DE-miRs and DEGs, and the overlapping miRNAs in cholestatic liver injury.
(A) Liver DE-miRs expression profiles between male Mdr2-/- and WT controls. (B) Liver DE-miRs expression profiles between female Mdr2-/- and WT controls. (C) Liver DEGs expression profiles between Mdr2-/- and WT controls. (D) The top 50 hug genes of the PPI network of upregulated DEGs and the top 50 hug genes of the PPI network of downregulated DEGs. (E) Predicted network of miRNAs – 100 identified hug genes of DEGs between Mdr2-/- and WT controls. (F) Identification of 11 overlapping upregulated miRNAs in cholestatic liver injury. FC, fold change; miR, micro RNA. DE, differentially expressed, Mdr2-/- , multidrug resistance 2 gene knockout; WT, wild type.

Fig 2. Exosome DE-miRs identification in Mdr2-/- mice compared to corresponding WT controls, the overlapping DE-miRs in the liver and serum exosomes, and comparison of the expression levels of these DE-miRs and target genes between normal and CHOL samples in TCGA CCA database.
(A) Exosome DE-miRs expression profiles between female Mdr2-/- and WT controls. (B) Identification of 2 overlapping upregulated DE-miRs in the serum exosomes which may contribute to cholestatic liver injury. (C) The expression levels of these 2 overlapping upregulated DE-miRs between normal and CHOL samples. (D) Heat map, the expression levels of these target hug genes of 2 overlapping upregulated DE-miRs between normal and CHOL samples.
Fibrosis development is associated with poor prognosis in the patient with chronic liver disease. The formation of bridging fibrosis (BF) is a critical step for developing advanced fibrosis and further cirrhosis. Preventing BF formation could be a strategy for inhibiting further advancement of fibrosis to cirrhosis. For that, underpinning the mechanisms of BF formation is to crucial. We previously showed Wilms tumor 1 (WT1) is a transcriptional regulator of profibrogenic HAS2 in liver fibrosis. Here, we further identified a novel profibrogenic function of WT1 in hepatic stellate cell (HSC) activation and invasion that is associated with BF formation in liver fibrosis.
First, we identified WT1 expression is upregulated in HSCs of BF area in advanced fibrosis in NAFLD patients, which is determined by our new experiment by combining scRNA-seq and spatial transcriptome analysis. Because HSC invasion to liver parenchyma and collagen production is critical for BF formation, we investigated the molecular mechanisms of HSC invasion using a collagen hydrogel invasion model in vitro. We performed RNA-seq for non-invaded HSCs and HSCs during and after invasion by PDGF treatment in vitro. During the invasion, HSCs quickly acquired the capacity to express WT1 and HSCs showed much higher proliferative and fibrogenic phenotype after invasion compared to HSCs without invasion. This invasive, proliferative, and fibrogenic HSC phenotype was inhibited by WT1 deletion. Expectedly, HSC-specific WT1 knockout mice showed reduced HSC invasion and BF development in murine liver fibrosis models induced by bile duct ligation (BDL) and choline-deficient high fat diet (CD-HFD) feeding, concluding WT1 is a potent pro-fibrogenic factor. Then, we investigated the regulatory mechanism of WT1 induction. We found WT1 promoter contains hypoxia inducible factor (HIF) binding site and hypoxic challenge is a strong inducer of WT1 and HSC invasion. Furthermore, our bioinformatics analysis revealed that WT1 is the most enriched transcription factor for hypoxia-induced invasive phenotype in HSCs. As the downstream effectors of WT1, our RNA-seq analysis by comparing wild-type and HSC-specific WT1 knockout mice found PDGFRB and Cathepsin K (CTSK). CTSK is also upregulated by WT1 overexpression in HSCs. Pharmacological inhibition of CTSK inhibited HSC activation in vitro and prevented BF formation in the BDL model. In conclusion, the WT1-CTSK-mediated profibrogenic actions in HSCs are crucial for HSC invasion and BF formation in liver fibrosis. Thus, targeting the WT1-CTSK axis to suppress invasive HSCs could be a novel strategy to prevent BF formation in patient with chronic liver disease.
