Effect Of Iron Overload On MiR-122 And Its Role In Liver Inflammation

BackgroundIron is an essential micronutrient, involved in many physiological process. But, the body can be damaged by iron overloaded. Iron overload in liver is find in many disease, such as nonalcoholic fatty liver disease, alcoholic liver disease, diabetes, hereditary hemochromatosis disease and the mediterranean anemia, which have mild or moderate inflammation. Resentely, dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute-on-chronic liver failure。 And some researcher have proposed tissue inflammation may be caused by hepatic iron overload. Although the damage of liver iron overload has received highly attention. However, the relationship between inflammation and iron overloaded in liver is still not clear.miR-122, an abundant liver-specific microRNA (miRNA), has been found its important role in liver inflammation and liver cancer. Recently, miR-122 plays an important role in regulating the expression of liver Hamp mRNA, which is the key regulatory factor of iron homeostasis in the body. But the connection between miR-122 expression and iron overload, as well as the role of miR-122 in liver inflammation caused by iron overload is unclear. Therefore, the subject is to study the effect of iron overload on miR-122 and its role in liver inflammation.ObjectiveThis study proposed by animal and cell experiment, molecular biology, biochemistry, liver pathological and inflammation score, to clarify the relationship between iron overload and liver inflammation; To study the effect of iron overload on miR-122 and confirmed that the role of miR-122 in liver inflammation caused by iron overload; To explore mechanism of liver inflammation caused by iron overload, providing new clues of prevention and treatment of liver inflammation caused by iron overload.Methods1. Animals1.1 puriftied diet group:C57 male mice, four weeks of age. Each weighing 13± 2 g, were purchased from Hayes Lake Company. They were feeding purified diet(purchased from Research Diets, Modified AIN-93GWith no Added Iron) and water freely.12 h/12 h rhythm,22± 2℃ After 3 days to adapt, they were divided into three sub groups:1 week group,2weeks group,4 weeks group, each divided into sub groups of iron overload, control, iron deficient. Control group mice were intraperitoneal injected with iron dextran (2mg/ml),20mg/kg, twice a week. Iron overload group mice were intraperitoneal injected with iron dextran (20mg/ml),200mg/kg, twice a week. Iron deficient mice were intraperitoneal injected with saline.1.2 Cereal Based diet group:Origin, gender, body weight in mice, raising environment were the same as purification diet group. After 3 days to adapt, they were divided into three sub groups:1 week group,2weeks group,4 weeks group, each divided into sub groups of iron overload, control. Mice were feeding cereal based diet, iron content is 200 mg/kg) of drinking water, according to the weight was divided into 2 groups:control group mice were intraperitoneal injected with saline. Iron overload group mice were intraperitoneal injected with iron dextran (20mg/ml),200mg/kg, twice a week. Mice were sacrificed in 7th,14th and 28th, serum and liver tissue were collected.1.3 Iron dextran solution preparation:20 ml/mg iron dextran solution was diluted by 100 mg/ml dextran iron 5 times (100 ml/mg iron dextran:physiological saline= 1 ml:4 ml, fully blending);2 ml/mg iron dextran was diluted by 20 ml/mg iron dextran 10 times (20 ml/mg iron dextran:physiological saline= 1 ml:9 ml, fully blending).2. Affymetrix Gene ChipLiver tissues were obtained from 15 mice(5 in each group), to detect the mRNA expression by the Gene expression profile chip (Affymetrix Gene Chip Mouse Gene 1.0 ST Array) with random variance model (RVM) for differences in Gene screening, analysis by MultiClassDif.3. Mice liver tissue Prussian blue staining, HE staining and inflammation scoreLiver samples were fixed in 4% buffered formalin and embedded in paraffin. After cutting 4-μm thick sections, all samples were stained with Prussian blue for iron deposition analysis or with hematoxylin and eosin (H&E) for histological observations, in Fluorescence microscope observation and collection of images.4. Real-time quantitative RT-PCRLiver tissues or cells of total RNA were extracted with Trizol, determination of its concentration, RNA was reversed-transcribed into cDNA using RT regent kit,miRNA used special RT Stem-loop primers. Mix cDNA template and primers, SYBR green, water blending, conducted on by Step One Plus real-time system. The animal mRNA levels was normalized to 18s of the sample. (cells:β-actin, miRNA:U6)5. Western-blot analysisKeyGene total protein kit was used to extract total protein of liver tissues or cells, determing the concentration of protein, degeneration, and SDS polyacrylamide gel electrophoresis, using the following antibodies:the CCL2 antibodies (1:400), p65 antibody (1:1000), IκBα antibody (1:1000), p-IκBa antibody (1:1000), TNFa (1:1000) and antibody (β-actin antibody,1:3000). Gray scale image analysis was normalized to P-actin.6. Liver tissue immunofluorescenceParaffin dewaxing, mice after antigen repair, add a resistance (CD68, TNFa, IL-1β), two resistance (including FITC appear bright yellow-green fluorescence), reoccupy DAPI stain nuclei (blu-ray) complex, with resistance to fluorescence quenching sealing seal tablets, confocal fluorescence microscopy and collection of images.7. Cell culture, intervention and TransfectionHuman liver cancer cell lines Huh7 cells, people stellate LX-2 cells were maintained in DMEM and supplemented with 10% fetal bovine serum (Gibigo) and 1% penicillin and streptomycin. The human monocyte cell line, THP-lcells can be differentiated into macrophages that mimic human macroph 5 age activity and function.Cells were maintained were maintained in RPMI 1640 and supplemented with 10% fetal bovine serum (Gibigo)and 1% penicillin and streptomycin under 5% CO2 at 37 ℃ in an incubator. Cells were then cultured in PMA (100 nM) in 24-well plates for 48 h to promote differentiation into macrophages. Cells were treated with FeSO4 or Holo-Tf added to the media for indicated time intervals.Huh7 cells were inoculated to plates. When alignment was 80%, transfect miR-122 mimics (miR-122 inhibitor, siRNA) with reagent lipofctamine RNAiMAX,37 ℃ incubation 15-20 minutes. Plasmid DNA transfection reagent is DNA transfection.8. Packaging virusAAV8 plasmid was extracted according to Calculates the quantity of the plasmid (http://www.geguchadze.com/calc), Send samples to Sangon company to identify the plasmid sequence, when HEK293 cells alignment was 80-90%, follow the steps and transfect the plasmid into cells. Collect and purification AAV8 virus cells, Detected degree of the virus by real-time quantitative RT-PCR.rAA8-CB-PI-pri-mir-122 GuassiaVirus and negative control virus were injected into the tail vein of IO mice(2 weeks) at 1×1011 vgs per animal, respectively. Mice were sacrificed in 28th, serum, liver, spleen, pancreas, lung, kidney, and brain were collected.9. Gaussia Luciferase AssayBlood samples were obtained, the supernatant was taken as blood serum (dilution 200 times)and the tissue protein of liver, spleen, pancreas, lung, kidney and brain was extracted.The Gaussia Luciferase was detected by Gaussia Luciferase Assay Kit.10. Luciferase reporter assaysThe wild-type human 3 ’UTR luciferase reporter vectors were constructed by amplifying CCL2 mRNA 3-UTR containing a potential binding sites and cloning it into p-MIR luciferase report plasmid vector cloning and binding sites of mutations to p-MIR luciferase report plasmid. Huh7 cells were co-transfected with the miR-122 mimics, after 48h the cells were collected for application in the Dual-Luciferase reporter system according to the manufacturer’s instructions11. Chromatin Immunoprecipitation (ChIP) AssayHuh7 cells were inoculated to 6cm2 plates, one group were treated by 30uM holo-transferrin, they ere negative control. Huh7 cells were cross-linked and processed according to the Millipore Chromatin Immunoprecipitation (ChIP) Assay Kit protocol.12. Stistical AnalysesStatistical techniques applied were the Student t test/Mann-Whitney’s U test and one-way analysis of variance or two-way analysis of variance. All statistical tests were two-tailed, and a significance level of P<0.05 was used P< 0.01 for the very significant level.Result1. Liver inflammation is related with iron overload.1.1 Animal experiment1.1.1 Gene chip results3282 genes have changed in iron overload mice detected by gene chip, in which Hamp, Ftl1 and Bmp6 significantly increase (P<0.001) while Trfc significantly decreases P (p<0.001). Besides, many inflammation-related factors such as TNF-a, CCL2, CCL3, CCR2, IL-1β, IL-6, CD68 and CD36 significantly increase (P<0.001). The results of real-time PCR detecting Hamp, Ftll, Bmp6, CCL2 and TNF-a are coincidence with gene chip. Furthermore, CCL2, p65, TNF-a, IL-6 and IL-1β significantly increase over time during 4 weeks iron overload mice detected by real-time PCR.1.1.2 Iron status in iron overload miceComparing to control group, serum iron, TS%, TIBC and hepatic iron increase over time in iron overload group fed by purified-diet or cereal based diet. Iron particles deposition can be found in mice liver by Prussian-blue staining.1.1.3 Changes of iron metabolism indicators and inflammation-related factors in iron overload liverCCL2, p65, TNF-a, IL-6 and IL-1β mRNA significantly increase over time during 1-4 weeks iron overload mice detected by real-time PCR.Protein expressions of TNF-α, IL-1β and CD68 increase in 2 and 4 week point iron overload mice liver detected by immunofluorescence. We also use western blot to detect protein expression of CCL2, p65, IkBα and p-IκBα, the results show that these inflammation-related factors highly increase.1.1.4 Liver HE staining and inflammation scores in iron overload miceThe inflammation cells and sore increase following the accumulation of iron.1.1.5 Serum biochemical indicators in iron overload miceIn iron overload mice, ALT and AST have no change in 1 week, but increase in 2 weeks and peak in 4 weeks.1.2 Cell experiment1.2.1 Effect of iron overload on cell activityThere are no difference of cell activity between treatment group and control group detected by flow cytometry.1.2.2 Effect of iron overload on iron amount in cellsThe content of iron in cells increases as the concentration of or FeSO4 and culture time increase.1.2.3 Effect of iron overload on expression changes of inflammation-related factors in Huh7, LX-2 cells, THP-1 derived macrophagesCCL2, p65, TNF-α, IL-6 and IL-1β mRNA significantly increase over time after treated by 30μM Holo-Tf in Huh7, LX-2 cells, THP-1 derived macrophages. CCL2, p65, IκBα, p-IκBα, TNFα protein significantly increase over time after treated by 30μM Holo-Tf in Huh7 cells. Also, CCL2, p65, TNF-a, IL-6 and IL-1β mRNA significantly increase over time after treated by 100μM FeSO4 in Huh7.2. Effect of iron overload on miR-122 and its role in liver inflammation2.1 Animal experiment2.1.1 Expression changes of mir-122 and pri-mir-122 in iron overload mice liverIn 1,2,4 weeks iron overload mice, miR-122 and pri-mir-122 expression in liver significantly decrease over time in purified-diet and cereal based diet group.2.4 Overespression miR-122 could partially alleviate liver inflammation caused by iron overload in vivo2.4.1 Guassia fluoresce expression in serum and main organs in rAAV8 injected miceThere are no difference in serum Guassia between control and treatment groups. And Guassia, carried by rAAV8, mainly expresses in liver while its content in other organs is very low.2.4.2 Expression of mir-122 in each groupmiR-122 expression in liver highly increases in miR-122 overexpression group with iron overload, while decreases in negative control group with iron overload and iron overload group.2.4.3 Changes of AST, ALT, liver iron, serum iron, TIBC and TS% in each groupIron overload mice with miR-122 overexpression in liver have normal level of AST and ALT, which is lower than iron overload group (P<0.05). But liver iron, serum iron, TIBC, TS% are still high (P<0.01). All these indicators increase in iron overload group and iron overload with negative control group (P<0.01).2.4.4 Expression changes of of inflammation-related factors in liver in each groupCompared to iron overload with negative control group, CCL2, p65,TNF-α, IL-1β and IL-6 mRNA expression in liver decrease in iron overload with miR-122 over expression group (P <0.001), but still higher than control group (P<0.05). These factors increase in iron overload group and iron overload with negative control group (P <0.001).Compared to iron overload with negative control group, protein expressions of CCL2, p65,TNF-α, IL-6 and CD68 in liver decrease in iron overload with mir-122 over expression group, but still higher than control group. These factors increase in iron overload group and iron overload with negative control group.2.2 Cell experiment2.2 Expression of mir-122 in cell linesmiR-122 is abundant in Huh7 cell but rarely expressed in HepG2 cells and THP-1 derived macrophages. So we take Huh7 cell for cell experiment.2.3 Effect of iron overload on expression of miR-122 in cellWe found that mir-122 begins to decrease with 30μM Holo-Tf and 100μM FeSO4, so we use this concentration for following experiments.2.4 Overespression miR-122 could partially alleviate liver inflammation caused by iron overload in vitroTransfect miR-122 mimics to huh7 and HepG2 cells can decrease the protein expression of CCL2, p65, IκBα, p-IκBα. TNFa, same as to transfect pri-mir-122 to HepG2 cells.We found that transfect Wild type CCL2 plasmid with mir-122 mimics can suppress expression of Luciferase Report Gene, while transfecting Mutant CCL2 plasmid with mir-122 mimics has no effect, which indicates that mir-122 can target to 3’UTR of CCL2.3. Mechanisms of miR-122 down-regulation induced by iron-overload3.1 Effect of iron overload on expression of pri-mir-122 expression in mice hepatic tissues and cellsThe experiments of mice and cells show that in the condition of iron overload, expression of pri-mir-122 in hepatic tissues a cells is significantly decreased, indicating that the iron inducing down-regulation of mir-122 in liver probably happened in transcription level3.2 Effect of iron overload on expression of transcription factor of miR-122 in cell experimentWe use real-time PCR to detect mRNA expression of HNF1α、HNF4α、HNF3β、 HNF6 and C/EBPa of huh7 cells in culture medium with 30uM holo-Tf and found only HNF4a is suppressed while other transcription factors have no significant difference.3.3 Effect of iron overload on expression of HNF4aBoth of mRNA and protein expressions of HNF4a are decreased in iron-overload mice and cells compared to control group. This result is coincide with changes of pri-mir-122 expression in hepatic tissues and cells with excess iron.3.4 Effect of suppress or overexpress HNF4a on expression of miR-122 expressionWe transfect HNF4asiRNA to hepatic cells to suppress HNF4a, and found that pri-mir-122 and miR-122 are significantly down-regulated while overexpress of HNF4a can up-regulate pri-mir-122 and miR-122.3.5 Effect of excess iron on binding activity between HNF4a and miR-122 promoter in hepatic cells.The binding activity between HNF4a and miR-122 promoter is weakened in the condition of iron overload detected by ChIP.Conclusion1. Iron overload could lead to liver CCL2, p65, TNF-a, IL-6 and IL-1β mRNA, inflammation score,AST and ALT significantly increase;2. Iron overload could lead to Huh7, LX-2 cells, THP-1 derived macrophages CCL2, p65, TNF-a, IL-6 and IL-1βmRNA significantly increase;3. Iron overload could the expression of chemokine the CCL2 by down regulate miR-122, activate the NF-kappa B, inducing or promoting inflammation of the liver. By effective measures to increase expression of miR-122, which was decreased in iron overload, could partially alleviate liver inflammation caused by iron overload;4. Iron overload may suppress the transcription factor HNF4 alpha to down-regulate the expression of miR-122.By animal and cell experiment, the study found:Iron overload could inhibit the expression of HNF4 alpha, which resulted in the reduction the expression of miR-122, and raised the expression of the CCL2, activate the NF-kappa B signaling pathways, inducing or promoting inflammation of the liver; and increase the expression of miR-122 may be one of effective ways to alleviate liver inflammation caused by iron overload.