The Effects Of SOX7 On Liver Fibrosis And Molecular Mechanism Based On Weighted Gene Co-expression Network Analysis

Background and Aims:Liver fibrosis(LF)is a pathophysiological response of liver tissue to chronic damage caused by different etiologies.Common causes include hepatitis virus infection,excessive alcohol consumption,biliary obstruction,nonalcoholic fatty liver disease(NAFLD),etc.Persistent LF can lead to liver structural damage,resulting in liver function injury and cirrhosis;ultimately leading to the occurrence of hepatocellular carcinoma(HCC)and liver failure.Hepatic stellate cells(HSCs)are considered the effector cells of the formation and resolution of LF.The activation of HSCs and their mediation of LF progression are regulated by various cytokines and signaling pathways.A comprehensive understanding of the mechanism of HSCs activation and the process of LF is essential for screening new targets for anti-fibrotic therapy.Weighted gene co-expression network analysis(WGCNA)is a systematic biological method used to describe gene correlation patterns among different samples.It can be used to identify gene sets with highly coordinated changes.The characteristics of modules are reflected by the characteristic genes or key genes in the modules.Through the correlation analysis between modules and phenotypes and clinical traits,core genes closely related to LF are identified,which could be important therapeutic targets and biological markers.The SRY-related high mobility group box 7(SOX7)is an important member of the SOX transcription factors family,which regulates cell proliferation,differentiation,and apoptosis.It plays roles in various developmental processes,such as the differentiation of endoderm,vasculogenesis,cardiovascular development,and the occurrence and development of various tumors.Previous studies have shown that SOX7 expression levels were reduced in many solid tumor tissues including HCC,serving as tumor suppressor.However,the expression changes and functions of SOX7 in HSCs activation and the process of LF are still unclear.Based on the above background,we combined differentially expressed genes(DEGs)analysis of HSCs with WGCNA of liver tissue using web-based microarray data to screen the transcription factors(TFs)closely related to LF and identify core transcription factor SOX7.Then experiments in vivo and in vitro were performed to explore the effect of SOX7 on HSCs activation and the process of LF and potential molecular mechanisms.Materials and methods:1.The two microarray data GSE68000 and GSE68001 of HSCs downloaded from the GEO database were combined and batch-corrected.Differential gene analysis was performed on quiescent and activated HSCs to screen out DEGs related to HSCs activation.The microarray data GSE89632 and GSE84044 of fibrotic liver tissue samples were combined and batch corrected for WGCNA.Then,the DEGs of HSCs data,the genes in fibrosis-related modules of fibrotic liver tissues data,and the human TF list were intersected to obtain TFs closely related to HSC activation and the process of LF.2.The expression levels of SOX7 in liver tissues of cirrhotic patients and controls were detected by immunofluorescence staining and qRT-PCR.Primary activated and quiescent HSCs were isolated from the liver tissues of cirrhotic patients and controls,respectively.The expression levels of SOX7 in the two groups of HSCs were detected by qRT-PCR.The LX-2 cells were stimulated by TGF-β1,and the expression changes of SOX7 before and after activation were detected by qRT-PCR and Western blot.The liver fibrotic mouse models induced by carbon tetrachloride(CCl4)and bile duct ligation(BDL)were constructed.The expression levels of SOX7 in mouse liver tissues were detected by immunofluorescence staining and qRT-PCR.3.The expression levels of SOX7 in LX-2 cells were reduced using a SOX7-specific si RNA.LX-2 cells were transduced by SOX7-specific lentivirus for stable SOX7 overexpression.The proliferation of LX-2 cells was detected by CCK-8,and cell apoptosis rates were detected by flow cytometry.The expression changes of SOX7 and protein levels of related signaling pathways were tested by Western blot.4.The C57BL/6J mice were tail intravenously injected with recombinant adenovirus encoding SOX7(AAV8-SOX7)to increase the expression levels of SOX7 in mouse liver tissues.Subsequently,the CCl4 induced mouse LF models were constructed.The expression changes of SOX7 and fibrotic genes in mouse liver tissues were detected by qRT-PCR,Western blot,and immunofluorescence staining.HE staining,Masson staining,and hydroxyproline(Hyp)content determination were performed on mouse livers to compare LF degrees.Results:1.A total of 3668 DEGs were screened out in activated HSCs(relative to quiescent HSCs),including 2079 up-regulated DEGs and 1589 down-regulated DEGs.Ten modules were generated in fibrotic liver tissue data after WGCNA.A total of 15 core TFs were screened out,among which HNF4 A,JUN,HES1,KLF5,and ID1 have been reported related to LF.To validate the expression changes of other selected TFs,qRT-PCR validation was performed.Of these TFs,SOX7,FOXQ1,HLX,SOX13,and ZNF511 exhibited consistent results in differential analysis and experimental verification.2.Compared with the control group,the transcription and protein levels of SOX7 were significantly reduced in liver tissues of cirrhotic patients.The transcription levels of SOX7 were also downregulated in the livers of CCl4 and BDL induced fibrotic mouse.The expression levels of SOX7 were reduced in activated HSCs induced by TGF-β1.3.SOX7 knockdown upregulated the expression levels of fibrosis marker genes COL1A1 and MMP2 in LX-2 cells and exhibited an increase in the proliferation capacity of HSCs and a decrease in apoptosis.SOX7 knockdown promoted the activation process of HSCs by enhancing the activity of signaling in the Wnt/β-catenin and the TGF-β/Smad,not the MAPK pathway.4.SOX7 overexpression downregulated the expression levels of COL1A1 and MMP2 in LX-2 cells,inhibited cell proliferation,and promoted apoptosis.SOX7 overexpression inhibited the activation of HSCs in response to fibrogenic stimulation by suppressing the Wnt/β-catenin and the TGF-β/Smad signaling pathways.5.SOX7 overexpression in vivo can alleviate the extent of CCl4 induced LF in mice,inhibit the expression of COL1A1 and ACTA2,and reduce the Hyp content in mouse liver tissues.Conclusion:1.By combining bioinformatic analysis with experimental validation,our results contribute to the understanding of the significant role of SOX7 in HSCs activation and the development of LF at both cellular and animal levels.2.SOX7 overexpression ameliorated the extent of LF along with inhibited HSCs activation and proliferation by suppressing TGF-β/Smad and Wnt/β-catenin pathways,which could be a promising therapeutic strategy for the prevention and treatment of LF.