Iron Overload Affects The Stability Of YY1 Protein Leading To Decreased Expression Of HNF4α

BackgroundIron is an essential trace element in the human body.Iron deficiency can cause anemia.However,iron overload can also cause great harm to the body.A large number of studies have confirmed that,as the main organ for iron storage in the body,liver iron accumulation can cause or aggravate liver tissue inflammation and lead to hepatocyte damage,which is related to liver fibrosis and even liver cancer.However,the mechanism by which iron overload causes inflammation has not been fully elucidated so far.In the past,most scholars believed that iron overload causes oxidative stress damage to hepatocytes and activates Kupffer cells to cause inflammation.However,studies have shown that antioxidant therapy can relieve oxidative stress caused by iron overload,but it can not reduce the inflammatory response.Interestingly,our previous study found that input iron overload can directly trigger the inflammatory response in liver tissue by inhibiting the activity of HNF4α/miR-122 signaling pathway.Hepatocyte nuclear factor 4αis a member of the nuclear receptor superfamily.HNF4αplays a role in the formation of liver during embryonic development,and hepatic lipid metabolism,drug metabolism and other normal physiological functions and liver inflammation,liver fibrosis and other pathological processes play a very important role,its decline in expression may be an important factor in the occurrence of hepatocellular carcinoma,overexpression of HNF4αcan significantly inhibit liver cancer and fibrosis,extended implantation The survival time of human cancer cell mice,overexpression of HNF4αin tumors can also slow the growth of tumors.However,no report has been made so far about how iron overload leads to a decrease in the expression of HNF4α.The elucidation of the molecular mechanisms underlying the decrease in HNF4αexpression caused by iron overload may be of great significance for understanding the effects of iron overload on liver tissue cells and iron overload related disease prevention and treatment measures.Therefore,this study aims to further understand the biological characteristics of HNF4αexpression changes caused by iron overload,and explore the molecular mechanism of iron overload inhibition of HNF4αexpression.ObjectiveThrough in vitro experiments to understand the biological characteristics of the decrease in HNF4αexpression caused by iron overload,clarify the molecular mechanism of iron overload caused by decreased expression of HNF4α,provide clues for more in-depth study of the effect of iron overload on liver tissue cells,and for the study of iron The prevention and treatment of overload-related liver diseases provide a theoretical basis.Methods1.Cell cultureHuman normal liver cell line L02 cells were cultured in 1640 medium containing 10%fetal bovine serum and 1%penicillin and streptomycin.Human hepatoma cell lines Huh7,G2 cells and mouse liver cancer cell line Hepa16 cells were cultured in high-glucoseDMEM medium containing 10%fetal bovine serum and 1%penicillin and streptomycin.According to experimental purposes,each group was incubated with Apo-Tf,Holo-Tf and FeSO4,respectively（5%CO2,37°C）.2.RT-PCRTotal RNA of cells and tissues was extracted with Trizol,the concentration was measured,and RNA was reverse transcribed into cDNA using a reverse transcription kit.After the cDNA was used as a template and mixed with primers,SYBR green,and water,amplification was performed on a StepOne Plus real-time quantitative PCR apparatus to obtain the results and statistical analysis was performed.3.Western blotTotal protein was extracted from the cells and tissues using a whole protein extraction kit,and the concentration was measured.After denaturation,the cells were subjected to SDS polyacrylamide gel electrophoresis,transferred to membrane,blocked,incubated with primary and secondary antibodies,and imaged and analyzed.4.Detection of intracellular iron contentThe cells were seeded in 12-well plates and cultured at 37°C until the degree of confluency was approximately 80%.Holo-Tf was added to the culture medium to a final concentration of 30 uM and cultured for 0.5 h.The medium containing Holo-Tf was removed,washed 3 times with an appropriate amount of PBS,added to PGFL（dissolve in DMSO）,the final concentration was 5 uM,incubated in the dark for 20 minutes,washed with 3 times of PBS,and the change of the green fluorescence intensity was observed under a confocal microscope.（wavelength 490nm,emission wavelength 520nm）.5.Methylation detectionThe L02 cells were plated in 24-well plates and divided into two groups,IO group and IO+5-Aza-CdR group.The IO group was added Holo-Tf at a final concentration of 30uM.The IO+5-Aza-CdR group was added in the same amount of Holo-Tf and 5-Aza-CdR at a final concentration of 2.5 uM.After cell intervention,they were collected at 0,1,2,4 and 8h in turn.The expression of HNF4αin each group was detected by real-time quantitative PCR.6.transcription factor binding activity chipUsing the transcription factor binding activity chip,to detect and screen the L02 cell transcription factor binding activity of the control group,Holo-Tf-1h（Holo-Tf intervention for 1 h）group,and HNF4αgroup（with addition of HNF4αfragment）.7.ChIPL02 cells were inoculated into 6 cm² dishes and divided into control group,Apo-Tf group and Holo-Tf group,and cultured for 1 hour.The experiment was performed according to the procedure of the CST ChIP protocol.8.Protein stability testingCycloheximide（CHX）can inhibits protein synthesis.We used CHX to intervene in control and iron-overloaded L02 cells and collected cells at 0,0.5,1,2,4,and 8 h to extract total protein.Western blot analysis was used to compare the changes in the expression of cell protein in the two groups and observe the stability of YY1 protein after iron overload.9.siRNA and overexpression plasmid transfectionL02 cells were inoculated on the culture plate.When the degree of fusion was 80%,transfection was performed according to the instructions.The siYY1 or YY1 overexpression plasmid and transfection reagent were mixed in a volume of 1:1 and allowed to stand for 15 min at room temperature,and then added to each group of culture medium.The cells were collected after 48-72 hours of culture.10.Experimental animal mice grouping and modeling methodsIron overload mice:Fifty male C57 mice were four weeks old.After 2 days of adaptation,they were randomly divided into 3 groups:control group,0.3%high iron feed group,and 3%high iron feed group.The control group and 0.3%high iron diet group were divided into 4 subgroups:1 month group,3 month group,6 month group and 12 month group.During the feeding,the mice freely drink water.After being fed for 1 month,3 months,6 months and12 months respectively,they were sacrificed.The serum and liver tissue specimens were collected for use.Adeno-associated virus mice:Forty male C57 mice were divided into eight groups:1)negative control group;2)YY1 interference group;3)overexpression YY1 group;4)feeding 3%high iron feed 1 month group;5)feeding 3%high iron feed 1 month+interference YY1 group;6)feeding3%high iron feed 1 month+overexpression YY1 group;7)feeding 0.3%high iron feed12 months group;8)feeding 0.3%high iron feed 12 months group+overexpressing YY1groupThe virus was injected through the tail vein and the injection volume was:NC virus:2×10¹¹ vgs;YY1 interference virus:2×10¹¹ vgs;over-expression of YY1 virus:2×10¹⁰ vgs.Each group of mice was sacrificed four weeks after tail vein injection and sampled.11.Detection of liver iron content in miceThe mouse liver digested liquid was brought to a volume of 10 ml,and the sample solution was introduced into a flame atomic absorption spectrometer.The absorbance was measured and the iron content of the sample was calculated.12.liver tissue Prussian blue staining,HE stainingLiver samples were fixed in 4%paraformaldehyde,embedded in paraffin,sectioned,then stained with Prussian blue,or stained with hematoxylin and eosin,and viewed under a microscope to collect images.13.Detection of serum biochemical indicatorsSerum ALT,AST,serum iron,total iron binding capacity,and transferrin saturation were all measured using a blood biochemical analyzer.14.Statistics and Analysis of DataExperimental data is expressed as（mean±standard deviation）.Wsternblot bands were analyzed using the Quantity One Image Analysis System;experimental data were analyzed and plotted using GraphPad Prism 7.00 software.Two independent sample t-tests and one-way analysis of variance（one-way ANVOA）were used for different types of data.Results were significant at P<0.05,and very significant at P<0.01.Results1.Iron Overload Down-regulates HNF4αExpression in Human Primary Liver Cells,Human and Mouse Hepatoma Cells1.1 Iron Intervention on Hepatocyte HNF4αmRNA Expression at Different TimeWe exposed human hepatic cell L02 and human hepatoma cell line Huh7 in culture medium supplemented with 30μM HOLO-Tf or 100 uM FeSO4 for 1 to 8 hours.The results showed that the exposure was 1 hour.HNF4αmRNA expression was significantly decreased.With prolonged exposure,there was no further significant decrease in HNF4αmRNA expression in both cells.1.2 The effect of iron intervention for 1 h on the expression of HNF4αmRNA and protein in various liver cell linesWe expanded the cell types,including human primary hepatocytes,human hepatocytes L02,human hepatoma cells Huh7,G2,and mouse hepatoma cells Hepa16 with 30uM.The culture medium of Holo-Tf was cultured for 1 hour.As a result,it was found that the expression of HNF4αmRNA and protein was significantly decreased in the above cells.1.3 Effect of Holo-Tf intervention on intracellular iron content for 0.5 hWe used PGFL（Phen Green FL）fluorescent dye on iron intervention cells were stained,the results show that using Holo-Tf intervention in human primary hepatocytes,humans Liver cells L02,human hepatoma cells Huh7,G2,and rat hepatoma cells Hepa160.5 h,compared with the control group,the intracellular fluorescence weakened,indicating that the intervention 0.5 h,iron has entered the cell;In addition,real-time quantitative PCR results also show that 30hM Holo-Tf intervention for 1h,the above-mentioned hepatocyte iron regulatory molecules expression corresponding changes in iron overload,that Hamp,FTL1 mRNA expression increased,TRFC mRNA expression decreased.1.4 The effect of iron intervention for 8 h on the expression of HNF4α,miR-122and inflammation-related factors in hepatocytesL02 and Hepal6 cells were cultured in 30uM Holo-Tf medium for 8 hours.RT-PCR results showed that the mRNA expression of HNF4αand miR-122 decreased significantly,and the expression of CCL2,IL6 and TNFa mRNAs of inflammatory related molecules increased.The expression of HNF4αand miR-122 mRNA in human liver cancer cell Huh7was significantly decreased,and the expression of CCL2,IL6,and TNFa mRNA in inflammatory related molecules was increased.2.Decreased binding activity of transcription factor YY1 and HNF4αgene binding site is an important cause of iron overload leading to decreased expression of HNF4α2.1 The effect of iron intervention on the expression of miR-34a in cells at different timeThrough bioinformatics analysis,miR-34a and HNF4αgene have binding sites. Therefore,we have exposed L02 cells in medium containing 30uM Holo-Tf for 0.5,1,2,4,and 8 h respectively.There was no significant change in the expression of miR-34a at the time point,suggesting that the decrease of HNF4αmRNA expression in hepatocytes may not be associated with miR-34a.2.2 The effect of iron overload on DNA methylation of HNF4αWe used 5-AZA-CdR to inhibit HNF4αDNA methylation and found out whether iron intervention affected the expression of HNF4αDNA methylation in L02 cells and thus affected its gene expression.The results showed that compared with the Holo-Tf alone group,Iron There was no significant change in the expression of HNF4αmRNA in the5-AZA-CdR group at the same time,suggesting that the decreased expression of iron overloaded HNF4αin L02 cells may not be related to the change of its DNA methylation level.2.3 The effect of iron overload on HNF4αtranscription factor2.3.1 Transcript binding and active chip detection We used a transcription factor-binding activity chip to detect changes in the binding activity of HNF4αtranscription factor in iron-overloaded hepatocytes.The results showed that there were 15 transcription factors with significant changes in the activity of Holo-Tf interfering with L02 cells for 1 h,of which 11 were decreased in activity（ratio greater than2 times）;the transcription factor decreased in probe signal after adding 1 kb upstream of HNF4α30,Holo-Tf activity decreased significantly after 1 h and NFE2,FAST-1（FOXH1）,and NFE2（YY1）were the transcription factors that weakened the probe signal;the three transcription factors and HNF4αpromoter region were predicted by software.Binding site.2.3.2 The effect of iron overload on the binding activity of YY1,FOXH1 and NFE2and HNF4αWe used ChIP assay to detect the change in the binding activity of HNF4αand the above three transcription factors after 1 h of iron intervention in L02 cells.The results showed that iron overloaded The binding activity of YY1,FOXH1,NFE2 and HNF4αall had an effect,among which YY1 activity was the most attenuated.2.3.3 Detection of abundance of three transcription factors in different hepatocyte cell linesWe detected the abundance of YY1,FOXH1,and NFE2 in different liver cell lines by RT-PCR.The results showed that,whether in L02,Huh7 or G2 cells,the expression of YY1 was significantly higher than the other two transcription factors.2.4 The effect of iron intervention on the expression of YY1 in L02 cells at different timeWe examined the expression changes of YY1 after iron intervention for different periods of time.The results showed that after 0.5,1,2,4 and 8 hours of iron intervention,the expression of YY1 mRNA did not change significantly compared with 0h,whereas the expression of YY1 protein decreased significantly from the 0.5h level and decreased further at 1h.After the basic remains unchanged.2.5 Iron overload on the stability of YY1 proteinWe added CHX（final concentration 0.6 uM）to the cell culture medium of the control and iron overload groups,and detected the protein expression levels at different time points by Western blot.The results showed that the YY1 protein in the iron overload group and the control group In comparison,the degradation rate increases significantly.It shows that the iron overload is caused by the decrease of the stability of YY1 protein and the increase of degradation,which leads to the decrease of protein expression.2.6 Effect of interference and overexpression of YY1 on the expression of HNF4αin vitroTo further clarify the regulation of YY1 on HNF4α,we constructed a small interfering RNA（siYY1）and YY1 overexpression plasmid targeting YY1.The expression of HNF4αwas observed by transfecting siYY1 and overexpression plasmids into L02 cells and iron overloaded L02 cells,and the results showed that the expression of HNF4αmRNA was also decreased when YY1 expression was inhibited,and the expression of HNF4αmRNA was increased when the corresponding YY1 was overexpressed.High,and overexpression of YY1 can also reverse the decline of HNF4αexpression caused by iron overload.3.Characteristics of liver YY1 expression in mice with different levels of iron overload and its effect on HNF4α/miR-122 signaling pathway3.1 Changes of YY1,HNF4α,miR-122 and inflammation in liver of mice with different degrees of iron overloadWe used the method of feeding 3%high iron feed for one month and feeding 0.3%high iron feed for 12 months respectively to make mouse iron overload model.3.1.1 Liver iron overload and changes of liver inflammation in mice fed with 3%high iron dietFeeding 3%high-iron feed in one month,liver iron content,serum iron,total iron binding capacity and transferrin saturation（%）were significantly increased,and iron overload was changed in iron-regulated molecules（Hamp,TRFC,FTL1）expression.The liver showed significant iron deposition;the expression of hepatic inflammatory factors was significantly increased,serum inflammatory factors,transaminases increased,CD68fluorescence increased,and inflammatory cells increased.3.1.2 Liver iron overload and changes of hepatic inflammation in mice fed 0.3%high iron dietFeeding 0.3%high iron feed to mice,liver iron content gradually increased from January;serum iron,total iron binding force and transferrin saturation（%）gradually increased from March,iron regulatory molecules(Hamp,TRFC,FTL1 Significant changes in iron overload,expression of hepatic inflammatory factors,and serum TNFa levels gradually increased;serum AST and IL6 levels began to increase in June;inflammatory cells in the liver increased;CD68 green fluorescence increased significantly;liver appeared in December.Obvious iron deposition.3.1.3 Expression changes of YY1,HNF4αand miR-122 in mouse liver with different degrees of iron overloadThe results of RT-PCR and Western-blot showed that compared with the control group,the liver miR-122 mRNA expression,HNF4αwere observed in mice fed with 3% high-iron diet for one month and those fed with 0.3%high-iron diet for months of March,June and December.The mRNA and protein expressions were significantly decreased; YY1 mRNA expression was not significantly changed in both groups of iron-overloaded mouse livers.In contrast,YY1 protein expression was significantly decreased in all groups of mouse livers.3.2 Effects of targeting liver overexpression or interference with YY1 expression on the expression of HNF4α,miR-122 and inflammation-related factors in mice with different degrees of iron overloadWe used AAV8 as a vector to target hepatic knockout and overexpression of YY1,and to analyze the role of YY1 in the liver HNF4α/miR-122 signaling pathway in different degrees of iron overload mice.3.2.1 Effect of Interfering with YY1 on the Expression of HNF4α,miR-122 and Inflammatory Related Factors in Mice LiverRT-PCR and WB results showed that compared with NC group,the expression of HNF4αand miR-122 in siYY1 group decreased,and the expression of CCL2,IL6 and TNFa increased.3.2.2 The effect of overexpression of YY1 on the expression of hepatic HNF4α,miR-122 and inflammation-related factors in miceRT-PCR and WB results showed that compared with the NC group,the expression of HNF4αand miR-122 in the oYY1 group was increased,and the expression of CCL2,IL6,and TNFa was decreased.The HNF4αin the liver of the 3%+oYY1 group of mice compared with the 3%group of mice.The expression of miR-122 was significantly increased,and the expression of CCL2,IL6 and TNFa was significantly decreased.The expression of HNF4αand miR-122 was also significantly increased in 0.3%+oYY1 mice compared with 0.3%mice,and the expression of CCL2,IL6 and TNFa was also significantly increased.decline.Conclusion1.Iron overload can downregulate the expression of HNF4αat the cellular level and the whole animal level;2,transcription factor YY1 can regulate the expression of HNF4αat the transcriptional level;3,iron overload can affect the stability of YY1 protein to reduce its protein,and HNF4αbinding activity weakened,leading to decreased expression of HNF4α;4.Overexpression of YY1 can relieve the inhibitory effect of iron overload on the expression of hepatic HNF4αin mice.