The Role And Underlying Mechanism Of Hepatocellular SOX9 In Liver Regeneration And Hepatocellular TGF-β Signaling Pathway In Liver Fibrosis

Part Ⅰ The role and underlying mechanism of hepatocellular SOX9 in liver regeneration[Background and Objective]The liver is an important parenchymal organ involved in metabolism and has a strong regenerative capacity.Partial hepatectomy is the main effective clinical treatment for liver diseases,such as liver trauma,liver abscess,benign mass lesions of the liver,and hepatocellular carcinoma.Small residual liver volume,hepatocyte inflammation,and lipid deposition will lead to serious complications,such as hypohepatia and hepatic failure.The regulatory mechanism of liver regeneration is of great significance in promoting postoperative liver tissue reconstruction and functional recovery and might provide new therapeutic targets.SOX9 controls the differentiation of multiple cells.In liver homeostasis,SOX9 is mainly expressed in choanocytes,and only a small amount of SOX9⁺/HNF4α⁺hepatocytes（"Hybrid hepatocyte"）exists in the periportal,which is involved in repairing chronic liver injury.Recent studies have shown that mature hepatocytes dedifferentiate to produce SOX9⁺ hepatocytes during chronic liver injury,which is an important source of neonatal hepatocytes in liver regeneration.However,the role of SOX9 in acute liver injury is still unclear.HNF4α is a member of the family of HNFs,which plays an important role in the maintenance of hepatocyte function.Studies have shown that hepatocyte-specific knockout HNF4a results in uncontrolled proliferation of hepatocytes after PHx surgery,leading to the death of mice.Previous studies have demonstrated that HNF4α transcriptionally regulates multiple microRNAs such as miR-124 and miR-381,and SOX9 is the target gene of miR-124 in lung cancer and melanoma.This study intends to explore the role and underlying mechanism of SOX9 in acute liver injury,and futher clarify the regulatory effect of HNF4α on SOX9.[Methods]1.To determine the role of SOX9 in liver regeneration1.1.The expression of SOX9 during the acute liver injuryThe model of acute liver injury was established by standard partial hepatectomy（sHx）or intraperitoneally injected with carbon tetrachloride（CCl₄）and the mouse liver tissues were collected at different time points to detect the mRNA and protein levels of SOX9.1.2.The effect of SOX9 on liver regeneration1.2.1.The Sox9^HKO and Sox9^f/f mice were analyzed for the ability of liver regeneration after sHx surgery or intraperitoneally injected with CCl₄.1.2.2.Sox9^HKO,Sox9^HOE mice,and control mice performed eHx surgery to construct the Small-for-size Syndrome（SFSS）model.The survival rate was used to evaluate the effect of SOX9 on SFSS.1.2.3.The ability of liver regeneration and hepatocyte proliferation was evaluated in the Sox9^HOE and Sox9^Ctrl mice after eHx surgery.2.To detect the mechanism of hepatocyte SOX9 promoting liver regeneration2.1.Sox9^HKO and Sox9^f/f mice were sacrificed after sHx surgery 48 hours,and the frozen liver tissues were prepared for sn-RNA-seq to detect the potential mechanism.2.2.Sox9^HKO and Sox9^f/f mice were sacrificed after sHx surgery 48 hours and isolated mouse primary hepatocytes were to extract RNA for RNA-seq.2.3.Hepatocytes were divided into SOX9⁺and SOX9-subsets according to the mRNA level of SOX9,and the differentially expressed cytokines between the two subsets were compared.The effect of SOX9 on the cytokine was clarified by regulating the expression of SOX9 in HepG2 cells.3.Clarify the mechanism of regulation on SOX9 by HNF4a3.1.The mice were sacrificed,and the liver tissues were collected at different time points after sHx surgery to detect the mRNA and protein expression of HNF4a in the liver.3.2.The expression of SOX9 of Hnf4αHKO mice and control mice were analyzed.In vitro,the expression of SOX9 was elevated by regulating the HNF4α expression in HepG2 cells to detect the regulatory relationship between SOX9 and HNF4α.3.3.Real-time PCR was used to detect the expressions of miR-124/381 after sHx in C57BL/6J mice,and the levels of miR-124/381 of Hnf4αHKO mice liver tissues.3.4.The level of SOX9 was elevated by regulating the miR-124/381 expression,and the luciferase reporter system was used to detect the effect of miR-124/3 81 on SOX93’UTR wild-type and mutant plasmids.3.5.HepG2 cells were infected with Ad-HNF4α to overexpress HNF4α and transfected with miR-124/381 inhibitor to inhibit the miR-124/381 to detect the mRNA and protein expression of SOX9.4.Statistical analysisMeasurement data subject to normal distribution are represented by X±SD,If the measurement data obey the normal distribution and the variance is homogeneous,the student’s t-test is used for comparison.If the measurement data do not obey the normal distribution or the variance is heterogeneous,the nonparametric test is used for comparison.P<0.05 indicates that the difference is statistically significant.[Results]1.SOX9 promotes liver regeneration1.1.The expression of SOX9 increased at the early stage and decreased at the later stage both in sHx and CCl₄ models.1.2.Hepatocyte-specific knockout of SOX9 inhibits liver regeneration:In the sHx and CCl₄ models,the liver weight/body weight rate and hepatocyte proliferation of Sox9^HKO mice decreased significantly compared with the control group.1.3.In the model of the SFSS,the survival of Sox9HK0 mice was decreased significantly compared to the control group,while the survival rate of Sox9^HOE mice was higher compared to the control group.1.4.Hepatocyte-specific overexpression of SOX9 promotes liver regeneration:In the eHx model,the liver weight/weight rate and the hepatocyte proliferation of Sox9^HOE mice were significantly increased compared to the control group after surgery.2.SOX9 transcriptional regulated TGF-αto promote hepatocyte proliferation2.1.The analysis of snRNA-seq showed that hepatocytes were divided into 10 cluster subsets,and the hepatocyte subsets were further clustered and annotated into 8 subsets（C0-C7）.Knockout of SOX9 in hepatocytes reduced the proportion of subsets C2-C7,and GSEA enrichment analysis showed that C4 and C7 were mainly in the cell cycle and cell proliferation-related signaling pathways.The dot plot showed that C4 and C7 showed high expression of Mki67 and Top2a,and the expression of Mki67 and Top2a in hepatocytes decreased after the knockout of SOX9.2.2.The KEGG enrichment analysis of hepatocyte RNA-seq differentially expressed genes showed that the knockout of SOX9 in hepatocytes down-regulated the cell cycle and up-regulated signaling pathways related to hepatocyte metabolism.GSEA analysis showed that the cell cycle was significantly down-regulated in Sox9^HKO mice.2.3.The cell chat analysis of snRNA-seq showed that the EGF signaling pathway was activated in hepatocytes,and the hepatocyte subsets secreted TGF-α.Furthermore,the expression of Tgf-α and Cxcl12 in the SOX9⁺ hepatocyte was higher than that of SOX9hepatocytes of Sox9^f/f mice,and the expression of was not significantly changed in the SOX9⁺hepatocyte and SOX9-hepatocytes of Sox9^HKO mice.2.4.TGF-α expression was upregulated by regulating SOX9 in HepG2 cells.ChIP-PCR demonstrated that SOX9 regulated the TGF-α by binding to the TGF-αpromoter,and luciferase reporter revealed that SOX9 overexpression increased the activity of the TGF-α promoter-reporter,but significantly weakened the activity of the mutant reporter.3.HNF4α inhibits the expression of SOX9 via miR-124/3813.1.The expression of HNF4α decreased in the early stage of liver injury and recovered to normal level after 7 days,which was negatively correlated with SOX9 expression level.3.2.The SOX9 expression increased in the hepatocytes of Hnf4αHKO mice.Overexpression of HNF4α in HepG2 cells decreased the expression of SOX9,while downregulated HNF4α increased the expression of SOX9.3.3.In sHx model,the expression of miR-124/381 decreased in the early stage and increased to a normal level in the later stage,and the expression of miR-124/381 was significantly inhibited in Hnf4αHKO mice.3.4.miR-124/381 inhibited the expression of SOX9 in HepG2 cells,and the luciferase assays showed the activity of SOX9 3 ’UTR wild-type reporter was decreased with upregulation of miR-124/381 expression,while the luciferase activity of the mutant reporter showed no significant effect.3.5.The level of SOX9 decreased by overexpression of HNF4α,which could be partially reversed by miR-124/381 inhibitor,indicating HNF4α inhibits the expression of SOX9 via miR-124/381.[Conclusion]1.The expression of SOX9 increased in the early stage of acute liver injury and decreased to normal microexpression at a later stage during liver regeneration;2.SOX9 promotes liver regeneration and improves the survival rate of the SFSS;3.Cell proliferation-related signaling pathway down-regulated with knockout of SOX9,and SOX9 regulates TGF-α expression by binding to TGF-α promoter transcription;4.HNF4α inhibits the expression of SOX9 via miR-124/381.Part Ⅱ The Effect and Mechanism of Hepatocytes TGF-βSignaling Pathways in Liver Fibrosis[Background and Objective]Liver fibrosis is characterized by the excessive deposition of extracellular matrix（ECM）in the liver,which occurs in response to chronic liver inflammation and the tissue repair process.The end stage of liver fibrosis often leads to distorted hepatic architecture,cirrhosis,hepatocellular carcinoma,and ultimately liver failure.Providing effective antifibrosis treatment at an early stage is important for preventing the progression of chronic liver disease.Liver fibrosis is a complex process involving many cytokines.Transforming growth factor-β（TGF-β）has been reported as a profibrotic cytokine,and strongly promotes liver fibrosis during chronic liver injury.Previous studies have demonstrated that TGF-βsignaling is involved in activating hepatic stellate cells（HSCs）during liver fibrosis.TGF-βactivates Smad-dependent and Smad-independent pathways by binding to its receptors on the cell membrane,including transforming growth factor β receptor 1（TβR1）and receptor 2（TβR2）.Previous studies have shown that blocking the TGF-β signaling pathway in HSCs effectively alleviates liver fibrosis.However,the effect of TGF-β signaling in hepatocytes on liver fibrosis remains controversial.In this study,we focus on the role and mechanism of the TGF-β signaling pathway in hepatocytes during the process of liver fibrosis by using the hepatocyte-specific knockout of Tgfbr2 mice to provide a new strategy for the clinical treatment of liver fibrosis.[Methods]1.To determine the effect of the TGF-β signaling pathway in hepatocytes on liver fibrosis1.1.The expression of Tgfbr2 in fibrotic liver tissues and primary hepatocytesMice were treated with CCl₄ to induce liver fibrosis.The mRNA and protein expression of Tgfbr2 in liver tissues and primary hepatocytes of the fibrotic mice and control mice were detected by Real-Time PCR and Western blotting.2.2 To clarify the effect of deletion of the TGF-β signaling pathway in hepatocytes on liver fibrosisTgfbr2HKO mice and control mice were obtained after treatment with AAV8-TBG-CRE or AAV8-TBG and treated with CCl₄ for 4 weeks to induce the fibrotic model.We sacrificed the mice and isolated the primary hepatocytes.To evaluate the level of liver fibrosis in the two groups,Real-Time PCR,western blotting,hydroxyproline content,and histopathological staining were performed.hepatocytes2.To detect the mechanism of alleviating liver fibrosis by hepatocytes-specific knockout of Tgfbr22.1.RNA was purified from the primary hepatocytes of Tgfbr2HKO mice and control mice and was used to perform the RNA-Seq analysis.2.2.Real-Time PCR,western blotting,and immunohistochemistry were used to verify the results of RNA-seq,including inflammatory markers,EMT-associated genes,and hepatic function genes.3.Statistical AnalysisPrism 8 software was used for statistical analysis and mapping.Two-tailed P<0.05 was considered statistically significant.Student’s t-test was used to analyze the data of experiments involving only two groups of equal variances.The unequal variances pairing using the Wilcoxon signed rank test or Mann-Whitney U test.P<0.05 of the difference has statistical significance.[Results]1.Knockout of Tgfbr2 in hepatocytes alleviates liver fibrosis in mice1.1.The expression of Tgfbr2 in hepatocytes increased in liver fibrosisTgfbr2 expression significantly increased in fibrotic livers and hepatocytes of mice treated with CCl₄ for two weeks and four weeks by Real-Time PCR and Western blotting.1.2.HE and Sirius red staining analyses showed that the deposition of ECM decreased in the liver of CCl₄-induced Tgfbr2HKO mice compared with Tgfbr2f/f mice.Compared with Tgfbr2f/f mice,the content of hydroxyproline in fibrotic livers was also decreased in Tgfbr2HKO mice.The downregulation of a-SMA（Acta2）and COL1A1（Collal）was confirmed by real-time PCR and western blot analysis.1.3.The liver function of Tgfbr2HKO mice after CCl₄ modeling was partially restored compared with that of Tgfbr2f/f mice.2.Hepatocyte-specific knockout of Tgfbr2 inhibits the inflammatory response and slows down the process of EMT2.1.RNA-seq analysis showed that the up-regulated genes induced by CCl₄ treatment were significantly decreased after Tgfbr2 knockout.KEGG analysis showed that the down-regulated genes were mainly enriched in inflammatory and immune response pathways.Consistently,real-time PCR confirmed that the expressions of these genes in primary hepatocytes and the results were consistent with RNA-seq.2.2.GSEA analysis showed that both Wnt and Notch signaling pathways,which promote epithelial-mesenchymal transition,were significantly decreased after Tgfbr2 knockout and EMT-associating genes up-regulated in hepatocytes of fibrotic livers that were repressed by Tgfbr2 depletion.Real-time PCR,Western blotting,and immunofluorescence staining verified RNA-seq results.3.Depletion of Tgfbr2 up-regulates the expression of hepatocyte nuclear factor and protects the hepatic function3.1.RNA-seq analysis showed that the down-regulated genes induced by CCl₄ injury could be partially restored by the knockout of Tgfbr2.KEGG and GSEA analysis confirmed that down-regulated genes in hepatocytes of Tgfbr2f/f fibrotic mice were mainly involved in metabolism pathways.Real-Time PCR of mouse primary hepatocytes confirmed the RNA-seq results.3.2.Real-time PCR,western blot,and immunohistochemistry staining also validated the alteration of Foxa1,Foxa2,Foxa3,and Hnf4α in fibrotic livers of Tgfbr2f/f mice and Tgfbr2HKO mice.[Conclusions]1.The expression of Tgfbr2 in hepatocytes increased in liver fibrosis;2.Knockout of Tgfbr2 in hepatocytes alleviates liver fibrosis in mice;3.Hepatocyte-specific knockout of Tgfbr2 inhibits the inflammatory response and slows down the process of EMT;4.Depletion of Tgfbr2 up-regulates the expression of hepatocyte nuclear factor and protects the hepatic function.