The Effect And Underlying Mechanism Of Histone Acetyltransferase Mof Deletion On Liver Function

Objective: To study the effect and underlying mechanism of histone acetyltransferase Mof deletion on liver function in embryonic and adult mice.Methods: 1.In order to study the expression level of Mof in liver injury,we collected the RNAseq data of various liver injury from GEO database.These RNA-seq data were analyzed by DESeq2 to identify differentially expressed genes,respectively.False discovery rate(FDR q-value)was calculated by adjusting P-values with the Benjamini-Hochberg method.Genes with FDR q-value<0.05 and Log2(Fold change)>1 were considered as DEGs.By analyzing and comparing the expression profile data of various liver injury tissues and normal liver tissues,we found that the expression level of Mof in NASH patients' liver tissues was significantly reduced in the RNA-Seq data(GSE134422 and GSE126848)from 18 NASH patients' liver tissues and 17 normal liver tissues.The NASH expression profile data were further analyzed for gene differential expression and KEGG pathway.DAVID Bioinformatics Resources 6.8 was used to analyze the KEGG pathway of specimen data,and the pathway corresponding to p<0.05 was considered to be meaningful.2.To study the role of Mof in mouse hepatocytes,we first selected transgenic mice Mof^f/f(Mof flox/flox)and albumin(Alb)-Cre mice for hybridization to obtain Mof^f/+;Alb-Cre hybrid mice.Further hybridization of heterozygous mice with this genotype to obtain offspring mice for gene identification.In this mouse model,Mof gene containing flox was knocked out in hepatic primordium at embryonic day 10.5(E10.5) when Cre was activated by Alb,which can be specifically expressed in embryonic liver parenchymal cells and hepatic precursor cells.The relationship between embryonic development and Mof specific knockout of embryonic liver cells was analyzed after Mof knock-out.PCR was used to identify the genotype of hybrid mice and embryos.The embryo shape of mice was observed and the effect of Mof on embryo development was detected by comparing the birth rate of mice with different genotypes.Hematoxylin-eosin(HE)staining and cytokeratin 8(CK8) immunofluorescence staining were used to detect the effects of Mof on embryonic liver structural development.In addition,we used adenovirus(AAV)to carry hepatocyte-specific thyroxin-binding globulin(TBG)promoter to activate Cre and specifically knock out the Mof gene in adult mouse hepatocytes to analyze the effect of hepatocyte-specific Mof knock-out on adult mouse liver.After DNA genotypes were identified by PCR,qRT-PCR was used to detect the change of Mof mRNA expression level,and Western blot was used to further verify the change of Mof and H4K16 ac protein levels after the knockout of Mof hepatocytes.HE staining and Sirius Red staining were used to detect the structural changes and fibrosis levels of liver in mice.Serum alanine aminotransferase(ALT)levels were measured to determine the effect of hepatocyte-specific Mof knockout on liver function in mice.3.To study the effect of Mof knockout not only in hepatocytes and but also Kupffer cells on liver,we obtained Mx1-Cre;Mof^f/f mice by mating Mx1-Cre mice with Mof^f/f mice.By abdominal injection of polyinosinic acid(Poly I : C)to activate Mx1-Cre,which can be specifically expressed in hepatocytes and Kupffer cells in liver,to knock out Moff/f genes in vivo.Western blot experiments of Mof and H4K16 ac were performed to detect protein level after Mof double knockout in these two kind of cell.Immunohistochemical staining of liver tissue by Mof antibody was further verified to show Mof knockout in liver parenchymal cells.The intracellular knockout of Mof was indirectly verified by immunofluorescence of CK8 and H4K16 ac cells.After Poly I : C injection,periodic observation of clinical symptoms and survival of Mof double knockout mice were recorded.HE staining and Masson's Trichrome staining were used to detect the structural changes and fibrosis levels of liver in mice.The effect of Mof double knockout on liver lipid metabolism was verified by oil red O staining.Mice biochemical function metabolism were verified by blood biochemical tests.RNA-Seq(n=3)analysis was performed on the liver of mice after Mof double knockout,and the differential genes and related KEGG pathways were analyzed and compared with the liver RNA-Seq(n=18)of NASH patients.4.To study the mechanism of liver inflammation induced by Mof double knockout in hepatocytes and Kupffer cells,we co-cultured different genotype of hepatocytes and Kupffer cells in transwell plate.Primary Mof^-/- hepatocytes were collected in AAV-TBG-Cre injected Mof^f/f mice.After collection of the culture medium of L929 cells which contain macrophage colony stimulating factor(MCSF),20 % this medium and 100 nM tamoxifen(4-OHT)were added into 10 % FBS IMDM medium to culture Mof^f/f;ER-Cre hematopoietic stem cells from mouse femur to obtain a large number of Mof^-/-macrophages.Mof double knockout models of hepatocytes and Kupffer cells induced by Mx1-Cre in vivo was simulated by Transwell co-culture of Mof^-/- primary hepatocytes and Mof^-/- macrophages.The coculture of Mof^-/- primary hepatocytes with Mof^f/f macrophages,and the co-culture of Mof^f/f primary hepatocytes with Mof^-/- macrophages simulated the Mof single knockout of hepatocytes and Kupffer cells in vivo,respectively.The apoptosis ratio of co-cultured hepatocytes was detected by flow cytometry PI and Annexin V staining.The protein expression level of apoptosis-related genes PARP,Caspase3 and cytochrome c in cytoplasm and mitochondria was detected by Western blot.qRT-PCR and Western blot were used to detect the mRNA and protein expression changes of CCL2,IL6,TNF?,INOS,TIMP and other genes in Mof^f/f and Mof^-/- macrophages,as well as the mRNA and protein expression changes of these genes after two days of stimulation with the conditional medium from cultured primary hepatocytes.Mof^f/f and Mof^-/- primary hepatocytes were cultured by sodium nitroprusside(SNP),an exogenous nitric oxide(NO)donor.Cell Titer-Glo assay was used to quantify ATP to detect the number of living cells in the medium.At the same time,mitochondrial red fluorescence probe was used to label mitochondria and observe mitochondrial morphological changes under confocal microscope.Results:1.The expression of Mof decreased in NASH liver tissue,and the liver fibrosis pathways in NASH patients were significantly activated and the metabolic pathways were inhibited.GEO data showed that compared with normal liver tissues,the expression of Mof in liver tissues of NASH patients was decreased(p<0.05).KEGG pathway analysis showed that the expression signals of cell collagen fibril organization,positive regulation of apoptotic process,inflammatory response and negative regulation of cell proliferation were significantly upregulated,while the expression signals of oxidation-reduction process,xenobiotic metabolic process,general metabolism and fatty acid metabolism were significantly down-regulated.Differential gene expression analysis showed that the expression of genes related to inflammatory response-related factors,mitochondrial apoptosis related,positive regulation of cell death and positive regulation of fibroblast proliferation were significantly increased,while the expression of genes related to metabolic pathways and oxidation-reduction process was significantly decreased.2.Mof deficiency in embryonic hepatocytes can induce embryonic death,but do not cause liver-related inflammation in adult mice.We obtained 53 offspring after hybridization between Mof^f/+;Alb-Cre hybrid mice.Out of 53 offspring,there were 31 Alb-Cre;Mof^f/+ and 18 Alb-Cre;Mof^+/+ pups.The ratio of Mof^f/+ to Mof^+/+ was statistically consistent with the 2:1 expected Mendelian ratio.However,only 4 Alb-Cre;Mof^f/f pups were born,which significantly differs from the expected 25% Mendelian ratio.Importantly,none of the liveborn Alb-Cre;Mof^f/f mice lived beyond day 15.These results suggest that complete Mof deletion in embryonic hepatocytes is perinatal lethal.Among the 53 offsprings from further hybridization of Alb-Cre;Mof^f/+ hybrid mice with the same genotype,we obtained Mof hybrid deletion,homozygous deletion and wild type mice.There were 31 Alb-Cre;Mof^f/+ and 18 Alb-Cre;Mof^+/+ pups.The ratio of Mof^f/+ to Mof^+/+ was statistically consistent with the 2:1 expected Mendelian ratio.However,only 4 Alb-Cre;Mof^f/f pups were born,which significantly differs from the expected 25% Mendelian ratio.Importantly,none of the liveborn Alb-Cre;Mof^f/f mice lived beyond day 15.However,in neonatal mice with Mof knockout,no significant changes in liver structure were detected by HE staining and CK8 immunofluorescence staining.These results suggest that complete Mof deletion in the liver is perinatal lethal,but do no induce liver fibrosis.After two weeks of AAV-TBG-Cre mediated Mof knockout in adult mice,qRT-PCR showed that the mRNA expression level of Mof in Mof knockout mice was significantly decreased,and Western blot showed that the protein expression level of Mof and its downstream molecule H4K16 ac was also significantly decreased.However,consistent with Mof specific knock-out in embryonic hepatocytes,in adult mice hepatocytes specific Mof knock-out model,the liver HE staining and Sirius red staining showed no changes in structure and fibrosis level.In addition,Pearson correlation analysis showed that there was no significant change in serum ALT level in mice(P>0.05).3.Hepatocytes and Kupffer cells Mof double knockout can cause liver inflammation,and its transcription has similar molecular characteristics with NASH patients' liver transcriptome.Within 60 days after Poly I:C activated Cre in the liver of Mof^f/f heterozygous mice to mediated Mof double knockout,all of these mice developed hepatitis and died(n = 87,p<0.001).After double knock-out,western blots of mouse liver showed that the protein expression levels of Mof and its downstream target H4K16 ac were also significantly decreased.Immunohistochemical staining of Mof showed that most of the cells except intrahepatic bile duct cells and endothelial staining became weaker after Mof double knockout.Cellular immunofluorescence of H4K16 ac showed that the fluorescence area of H4K16 ac in the cells was significantly reduced under the condition of CK8 unchanged.HE staining showed that the liver tissue structure was disordered.Masson's Trichrome staining showed that blue fibrosis was significantly increased.Oil red O staining showed that small red lipid droplets were accumulated.Blood biochemical tests showed that liver injury indexes alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were significantly increased.Similar to the liver RNA-Seq data of NASH patients,KEGG pathway analysis of liver RNA-Seq data of Mof double knockout mice showed that negative regulation of cell proliferation,apoptotic process,response to lipopolysaccharide,cytokine response,and fibroblast proliferation were significantly up-regulated.Interestingly,the enriched gene pathways for the down-regulated genes almost exclusively involve metabolic processes,such as oxidation reduction and the sterol and cholesterol biosynthetic pathways.By comparing and analyzing the differentially expressed genes in the mice liver RNA-Seq expression profile with NASH patients GEO data,it was found that 264 differentially expressed genes were coincident,in which 62.5 % of the gene mRNA expression level increased at the same time,and 16.29 % of the gene expression decreased.In addition,after comparing RNA-Seq differential genes related pathways in human NASH and the Mof deletion mouse model,we find that pathways were highly overlapped in inflammatory factors,mitochondrial apoptosis and programmed cell death.4.Mof^-/- Primary hepatocytes can stimulate Mof^-/- macrophages to secrete a large number of liver inflammatory factors which cause mitochondrial damage and apoptosis of hepatocytes.After co-culture with Mof^-/- primary hepatocytes and Mof^-/- macrophages,apoptosis of primary hepatocytes was significantly increased by flow cytometry PI and Annexin V staining testing.Western blots analysis showed that the expression levels of apoptosis related genes PARP and Caspase3 were significantly increased,and the protein expression level of cytochrome c,gene related to mitochondrial damage in hepatocytes,was also significantly increased in cytoplasm.After Mof knockout in macrophage,qRT-PCR and Western blot analysis showed that the mRNA and protein expression of genes,such as CCL2,IL6,TNF?,INOS and TIMP related to hepatitis,did not change significantly.However,after two days of stimulation with the conditioned medium from cultured Mof^-/- primary hepatocytes,but the mRNA and protein expression of these genes in macrophages were significantly increased.Among thses fibrosis genes,fold change of INOS gene was the most obvious.Importantly,heat treatment of the Mof^-/- primary hepatocytes conditioned medium abolished its ability to activate Mof^-/- macrophages.Under exogenous NO stimulation from SNP,Cell Titer Glo assay showed that ATP quantitative value of Mof^-/- primary hepatocytes was significantly lower than that of Mof^f/f primary hepatocytes.Under the confocal microscope,the mitochondria of Mof^-/- primary hepatocytes were significantly damaged and dotted,while the mitochondria of Mof^f/f primary hepatocytes were relatively normal and linear.Conclusion: The expression of Mof was decreased in NASH liver tissue.Hepatocytes Mof deficiency can induce embryo death,but do not cause liver-related inflammation in mice.Hepatocytes and Kupffer cells Mof double knockout can cause liver inflammation,and its transcription has similar molecular characteristics with NASH patients' liver transcriptome.Mof^-/- primary hepatocytes can stimulate Mof^-/- macrophages to secrete a large number of liver inflammatory factors which can induce mitochondrial damage and apoptosis.