| Objective:Hepatic fibrosis is a widespread basic liver disease with poor prognosis,which often leads to decompensation of cirrhosis and hepatocellular carcinoma.Liver injury could activate the wound healing process,resulting in the production and accumulation of fibrous scar tissue in the intercellular substance.In resting state,extracellular matrix（ECM）can continuously maintain production and degradation,and maintain dynamic balance.When the balance of ECM deposition and clearance is broken,the pathogenesis of hepatic fibrosis begins.Liver injury could directly lead to the death of hepatocytes（HC）,which in turn release chemokines and recruit inflammatory cells to the injury site.Apoptosis and necrotic HC together with immune cells release TGF-βand other inflammatory cytokines,to activate resting nonproliferation hepatic stellate cells（q HSC）and derive into contractile myofibroblast hepatic stellate cells（a HSC）with activation state,in the process producing a large amount of collagen.The activation process of HSC can be regarded as a part of the physiological wound healing reaction of liver injury.However,overactivation and proliferation of a HSC with long term are pathological.a HSC not only secrete ECM,but also produce inflammatory cytokines and growth factors relying on autocrine and paracrine action,interacting with HC,immune cells and myofibroblasts derived from other cell lineages.The changed ECM could react on HSC and further regulate the activation and proliferation of HSC.The forkhead box（FOX）gene family was originally discovered in Drosophila as a genetic mutant.The forkhead domain is an important feature of FOX family proteins,forming a‘wing-like helix’structure via threeα-helices,threeβ-sheets,and a two-sided loop,with a highly conserved DNA sequence containing 110nucleotides.FOXO family is widely expressed in cells as a subfamily of the transcription factor FOX.FOXO4 protein could be regulated bilaterally through phosphorylation,acetylation and other modifications,which plays a key role in the biological function.YH0618 promoted viability and inhibited DOX（Doxorubicin）-induced apoptosis of normal hepatocytes L-02 cells through a mitochondrial-dependent mechanism mediated by FOXO4,and it reduced the toxicity caused by DOX.Isoorientin inhibites AKT phosphorylation and increases FOXO4 expression through mitochondrial dysfunction and the PI3K/AKT signaling pathway in cells,thereby inducing cell death in a concentration-dependent manner.This process was not toxic to normal liver cells.HBV could promote mi R-328-3p expression through the STAT3 signaling pathway and downregulate its target,FOXO4,resulting in cell damage to THLE-2 hepatocytes infected with HBV.upregulation of FOXO4 expression mediated by HBX enhanced resistance to cell death induced by oxidative stress.HBX is a regulatory protein of HBV and produces ROS in human liver cell lines.HBX induces FOXO4 upregulation in Chang cells that stably express HBX（Change-HBX cells）and in primary liver tissues from HBX transgenic mice.However,the expression and specific mechanism involved in the pathogenesis and development of hepatic fibrosis of FOXO4 in HSC and HC is still unclear.Methods:1.The research group collected the tissue samples from the patients with hepatocellular carcinoma（hepatocellular carcinoma tissues,hepatic fibrosis tissues adjacent to cancer tissues）and the tissue samples from the patients with hepatic hemangioma（normal liver tissues adjacent to hepatic hemangioma）and liver surgical trauma.Western Blot and q RT-PCR methods were used to detect the expression of FOXO4 protein in 20 groups of hepatocellular carcinoma tissues,para-cancer hepatic fibrosis tissues and randomly matched normal liver tissues.IHC method was used to detect the expression and protein localization of FOXO4 in 20groups of para-cancer hepatic fibrosis tissues and randomly matched normal liver tissues adjacent to hepatic hemangioma or resection during liver surgical trauma.Western Blot and q RT-PCR methods were used to detect the expression of FOXO4protein and m RNA in human hepatic stellate cells（HSC）LX-2 cells,human hepatocytes（HC）THLE-2 cells and two kinds of cells induced by TGF-βwith activation state,and in several human hepatocellular carcinoma cell lines（Hep-G2,Huh-7,MHCC97-H）.The mouse model of hepatic fibrosis was established by intraperitoneal injection of CCl4dissolved in olive oil into BALB/c mice.After 8weeks of modeling,the liver tissues were obtained.After HE staining,Masson staining and the test of hydroxyproline in the liver tissues,the test of ALT and AST in serum were measured to determine the formation of the model.Western Blot,q RT-PCR and IHC methods were used to detect Foxo4 and several fibrosis biomarkers（α-SMA,Col1,Mmp2,Ctgf,Timp1）.The primary HSC and HC of mice were extracted,and the extracted cells were identified by IF,Periodic Acid-Schiff staining and electron microscope observation.Western Blot and q RT-PCR methods were used to detect Foxo4 and several fibrosis biomarkers（α-SMA,Col1）in primary HSC and primary HC extracted from model group and control group respectively.In addition,the primary HSC of BALB/c mice with normal growth were extracted,and Foxo4 and several fibrosis biomarkers（α-SMA,Col1）in the primary HSCs immediately attached to the culture dish after extraction and cultured in vitro for 7 days were detected by Western Blot and q RT-PCR methods respectively.2.Different concentrations or different stimulation time of TGF-βwas used to stimulate human HSC cells（LX-2）and human HC cells（THLE-2）,Western Blot and q RT-PCR methods were used to detect FOXO4 and several fibrosis biomarkers（α-SMA,Col1）.Gene transcription inhibitor:Actinomycin D,protein translation inhibitor:Cycloheximide,proteasome inhibitor:MG132 were interfered with TGF-βactivated LX-2 and THLE-2 cells.Western Blot and q RT-PCR methods were used to detect FOXO4 protein.TGF-βstimulated for a short time with LX-2 and THLE-2cells,Western Blot method was used to detect p-FOXO4（phospho S193）with the expression of FOXO4 protein as the internal reference.In addition,through separation and extraction of nucleus and cytoplasm protein,Western Blot method was used to detect FOXO4 protein in nucleus and cytoplasm of at different time points.IF method was used to observe the localization of FOXO4 protein in LX-2and THLE-2 cells stimulated by TGF-β.Human FOXO4 silencing sh RNA plasmid was designed and human FOXO4 silencing sh RNA lentivirus was also designed in this way.Human FOXO4 overexpression plasmid was designed.These vectors were transfected into LX-2 and THLE-2 cells,and Western Blot and q RT-PCR methods were used to verify transfection efficiency.LX-2 cells transfected with FOXO4silencing sh RNA lentivirus and its NC lentivirus stimulated by TGF-β,Western Blot and q RT-PCR methods were used to detect the expression of several fibrosis biomarkers（α-SMA,COL1,MMP2,CTGF,TIMP1）,several proliferation biomarkers（Cyclin D1,PCNA,Ki67）of LX-2 and several apoptosis biomarkers（Bax,Bad）of THLE-2 according to the regulation of FOXO4.Plate clone formation and CCK8 methods were used to detect the capacity of proliferation of LX-2.Flow cytometry method was used to detect the capacity of apoptosis of THLE-2.LX-2transfected with silencing sh FOXO4 lentivirus and its sh NC were used to screen key targets with the downstream of FOXO4.Western Blot and q RT-PCR methods were used to detect the these key targets in LX-2 and THLE-2 cells according to the regulation of FOXO4.3.The key targets COL1A2 and KDM4A were selected according to the results of m RNA sequencing.The base expression in the region of the TSS of the proximal promoter of human COL1A2 gene-378bp to+58bp was queried through NCBI website.The JASPAR database was used for bioinformatics prediction and analysis to obtain the potential binding sites associated with the sequence.CHIP-q PCR method was used to detect the ability of recruiting transcription factor FOXO4 at-32bp to-26bp of COL1A2 gene upstream TSS in LX-2 and THLE-2 cells,and to compare recruitment ability stimulated by TGF-βin LX-2 and THLE-2 cells.COL1A2 proximal promoter p GL3 plasmid and its binding site mutant plasmid were designed,and double luciferase reporter gene method was used to detect the changing of the luciferase activity of COL1A2 gene caused by stimulation of TGF-βor overexpression of FOXO4,and compared with the mutation of the binding site.Co-IP method was used to detect the binding ability of FOXO4 and KDM4A proteins in LX-2 and THLE-2 cells.The binding of FOXO4 and KDM4A in LX-2and THLE-2 cells was detected by IF method.Ch IP-q PCR method was used to detect the recruitment of KDM4A and H3K9me3 near the binding region of FOXO4and COL1A2 proximal promoter.Ch IP Re-IP method was used to detect the ability of FOXO4 and KDM4A to combine with each other and bind to the proximal promoter of COL1A2 gene in the region of the binding site of COL1A2 proximal promoter.LX-2 and THLE-2 cells were interfered by ML324 through stimulation of TGF-βor overexpression of FOXO4.r AAV8-Foxo4 was injected into the tail vein of the hepatic fibrosis mouse model,and the expression of Foxo4 in the liver was silenced in vivo.After HE staining,Masson staining and the test of hydroxyproline in the liver tissues,the test of ALT and AST in serum were measured to determine the degree of hepatic fibrosis after the change of the expression of FOXO4.Western Blot,q RT-PCR and IHC methods were used to detect Foxo4,the key targets and several fibrosis biomarkers（α-SMA,Col1a1,Col1a2）of liver tissue and primary HSC,HC.Results:1.The expression of FOXO4 protein in 20 hepatic fibrosis tissues was significantly increased,and the expression of FOXO4 in HCC tissues was also significantly higher than that in normal liver tissues.But there was no significant difference between hepatic fibrosis tissues and HCC tissues.The m RNA of FOXO4in the 20 groups tissues of patients expressed the same content.20 cases of hepatic fibrosis tissues all showed FOXO4 protein positive in IHC method.FOXO4 protein was located in the cytoplasm and nucleus,with more expression in the nucleus.But20 cases of normal liver tissues was all negative,and FOXO4 protein was basically expressed in the cytoplasm.The protein and m RNA levels of FOXO4 in activated LX-2 and THLE-2 cells were significantly higher than those in cells at rest.The levels of FOXO4 protein and m RNA in multiple HCC cells（Hep G2,Huh-7,MHCC97-H）were also significantly higher than those in LX-2 and THLE-2 cells at rest.The mouse model of hepatic fibrosis was established after 8 weeks of modeling.HE staining,Masson staining and the test of hydroxyproline in the liver tissues,the test of ALT and AST in serum were measured to determine the formation of the model.The expression of Foxo4 protein and m RNA in the liver of mice with hepatic fibrosis model increased,and the Foxo4 protein mainly expressed in the nucleus,while Foxo4 in the liver tissue of the control group is mainly expressed in the cytoplasm.The primary HSC and HC of mice were extracted,and the extracted cells were identified by IF,Periodic Acid-Schiff staining and electron microscope observation.The expression of Foxo4 in the primary HSC and HC extracted from the model group mice was significantly increased,and the expression of Foxo4 was increased after the primary HSC was cultured and activated for 7 days in vitro.2.LX-2 and THLE-2 cells were treated with different concentrations of TGF-β（0-20ng/ml）and different stimulation time（0-48h）,and the expression of FOXO4gradually increased in a concentration and time dependent manner.The expression of FOXO4 increased sharply at 10ng/ml,24h under TGF-βstimulation.Actinomycin D and cycloheximide reduced the expression of FOXO4 protein in LX-2 and THLE-2 cells,but MG132 did not have such effect.LX-2 and THLE-2cells stimulated by TGF-βfor a short time（0-8h）,the expression of FOXO4 did not change significantly,while the expression of p-FOXO4（phospho S193 site）protein decreased.During this process,the expression of FOXO4 in the nucleus increased gradually.Nuclear localization of FOXO4 protein in LX-2 and THLE-2 cells increased stimulated by TGF-β.The vectors were transfected into LX-2 and THLE-2 cells,and transfection efficiency met the experimental requirements.The regulation of FOXO4 could reduce or increase several fibrosis biomarkers（α-SMA,COL1,MMP2,CTGF,TIMP1）.The regulation of FOXO4 in LX-2 cells could affect the expression of several proliferation biomarkers（Cyclin D1,PCNA,Ki67）.Both plate clone formation and CCK8 methods proved that the regulation of FOXO4 could affect the proliferation ability of LX-2 cells.The regulation of FOXO4 in THLE-2 cells could affect the expression of several apoptosis biomarkers（Bax,Bad）.Flow cytometry method proved that the regulation of FOXO4 could affect the apoptosis ability of THLE-2 cells.The m RNA sequencing screened the key targets COL1A2,KDM4A.The expression of KDM4A increased,and the expression of H3K9me3 decreased in LX-2 and THLE-2 cells stimulated by TGF-β.3.The JASPAR database was used for bioinformatics prediction and analysis.It was found that the upstream-32bp to-26bp in proximal promoter region of the TSS of COL1A2 gene was the potential binding site of the transcription factor FOXO4.Ch IP method proved that FOXO4 could bind to the-32bp to-26bp region,while the recruitment ability increased stimulated by TGF-β.Double luciferase reporter gene method found that the activity of COL1A2 gene caused by stimulation of TGF-βand FOXO4 overexpression could significantly enhanced,and this activity could be completely inhibited after mutation of this site.Co-IP method and IF method found that FOXO4 protein and KDM4A protein could bind each other in LX-2 and THLE-2 cells,while the binding ability increased stimulated by TGF-β.Ch IP method proved that there was recruitment of KDM4A and H3K9me3 near the binding region of FOXO4 and COL1A2 proximal promoter,while the recruitment ability increased stimulated by TGF-β.The recruitment ability of KDM4A in LX-2and THLE-2 cells was significantly increased,and the recruitment ability of H3K9me3 was significantly decreased stimulated by TGF-β.In addition,the situation was in contrast during the FOXO4 decreasing.Ch IP Re-IP method found that FOXO4 and KDM4A in the COL1A2 proximal promoter region combined with each other and combined to bind in the gene proximal promoter,while the recruitment ability increased stimulated by TGF-β.This would lead to the decrease of COL1A2 transcription activity.The decrease of KDM4A induced by ML324could significantly inhibited the expresssion of fibrosis biomarkers LX-2 and THLE-2 cells induced by stimulation of TGF-βor overexpression of FOXO4.Silencing r AAV8-Foxo4 was injected into the tail vein of the hepatic fibrosis mouse model,and the expression of Foxo4 in the liver was silenced in vivo.HE staining,Masson staining showed that the degree of hepatic fibrosis relieved,and the level of hydroxyproline in the liver tissues,the level of ALT and AST in serum decreased.The expression of several fibrosis biomarkers（α-SMA,COL1A1,COL1A2）decreased.With the decrease of Foxo4 expression in mouse liver tissue,the expression of Kdm4a in decreased,and the expression level of H3K9me3 increased.The primary HSC and HC extracted from mice in each group also showed the same content.Conclusion:1.The expression of FOXO4 increased in hepatic fibrosis tissues and cells,and increased in HCC tissues and cells.But there was no significant difference in the expression of FOXO4 in hepatic fibrosis and HCC tissues.FOXO4was mainly expressed in the nucleus of liver fibrosis tissues,and in the cytoplasm of normal liver tissues.2.After treatment with TGF-βof liver cells,the transcription and translation of FOXO4 were increased.During the process,FOXO4 protein was dephosphorylated（pho S193 site）and transferred into the nucleus.FOXO4 promotes the activation and proliferation of hepatic stellate cells（HSC）.FOXO4 promotes fibrosis and apoptosis of hepatocytes（HC）.3.TGF-βmediated the increase of FOXO4,and activated COL1A2 expression by relying on the transcription pathway induced by the interaction with KDM4A and the recruitment in the COL1A2 proximal promoter of its demethylated label H3K9me3.ML324,a KDM4A-specific inhibitor,had the potential to alleviate the pathogenesis of liver fibrosis.In vivo animal experiments confirmed that FOXO4intrahepatic knockdown could slow down the pathogenesis of hepatic fibrosis. |
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