MiR-125b Promotes The NF-κB-mediated Inflammatory Response In NAFLD Via Directly Targeting TNFAIP3

Non-alcoholic fatty liver disease(NAFLD)is one of the most common liver diseases in the world.The increasing prevalence and incidence of NAFLD have brought a huge medical and economic burden to the world.Without timely intervention and treatment,NAFLD may progress from early non-alcoholic steatosis(also known as simple fatty liver)to non-alcoholic steatohepatitis(NASH),liver cirrhosis,hepatocellular carcinoma(HCC),and even death.Currently,there is no effective pharmacological therapies are approved for the treatment of NAFLD,and a thorough understanding of the pathogenesis of NAFLD is essential for the development and establishment of new treatment strategies.miR-125 b is abnormally highly expressed in NAFLD.Target Scan predicts that TNFAIP3 may be a downstream target of miR-125 b.Although miR-125 b is closely related to the occurrence of liver fibrosis and HCC,but the exact role of miR-125 b and TNFAIP3 in the pathogenesis and disease progression of NAFLD is still unknown.miRNA is a kind of endogenous non-coding small RNA,which includes approximately 14–24 nucleotides in length.miRNA can bind and degrade the m RNA level of target gene or inhibit the translation process by base complementary pairing with the 3’ terminal untranslated region(3’ UTR)of the m RNA molecule,and participate in a variety of physiological and pathological processes by regulating the expression level of the target gene.Several studies have shown that a variety of miRNAs are involved in regulating the development of fatty liver and liver fibrosis,of which miR-125 b is a miRNA abnormally highly expressed in NAFLD,and miR-125 b is closely related to the development of liver fibrosis and HCC.The pathological changes of NAFLD mainly include hepatocyte steatosis,necrosis and hepatocyte apoptosis,which leads to the development of steatohepatitis,liver fibrosis and cirrhosis.NF-κB signaling pathway is a key factor mediating the inflammatory response and can be involved in the pathogenesis and disease progression of NAFLD through a variety of mechanisms.Studies have found that tumor necrosis factor-induced protein 3(TNFAIP3)is a key negative regulator in the pathological process of NAFLD.Target Scan predicts that TNFAIP3 may be a downstream target of miR-125 b.The exact role of miR-125 b and TNFAIP3 in the occurrence and disease progression of NAFLD remains unknown.Part One Correlation between miR-125 b and TNFAIP3 in patients with NAFLDObjective: To explore the expression levels of miR-125 b and TNFAIP3 in patients with NAFLD and the correlation between them.Methods: Peripheral blood samples were collected from patients with NAFLD and healthy volunteers.The levels of TNF-α,IL-6 and IL-1β in peripheral blood were measured by ELISA.The expression levels of TNFAIP3 and miR-125 b in peripheral blood samples were detected by RT-PCR.The relationship between TNFAIP3 and miR-125 b was predicted and analyzed by Target Scan.Results: The secretion of TNF-α,IL-6 and IL-1β in the peripheral blood of NAFLD patients were significantly up-regulated,the expression level of miR-125 b was significantly increased,while the expression level of TNFAIP3 was significantly decreased,and miR-125 b had a binding site with the 3’ UTR region of TNFAIP3.Conclusion: The inflammatory response is exacerbated in NAFLD patients,miR-125 b is positively correlated with the occurrence and development of NAFLD,while TNFAIP3 is negatively correlated with the occurrence and development of NAFLD.TNFAIP3 may be a direct downstream target of miR-125 b.Part Two TNFAIP3 affects the inflammatory response in cell model of NAFLD by regulating the NF-κB signaling pathwayObjective: To explore the regulation of TNFAIP3 on inflammatory response in the development of NAFLD.Methods: Hep G2 cells were treated with free fatty acid(FFA)to construct cell model of NAFLD.The expression level of TNFAIP3 was detected by RT-PCR.The level of TNFAIP3 in NAFLD cell model was knocked down or overexpressed by cell transfection technique.The expressions of TNF-α,IL-6 and IL-1β in different groups were detected by ELISA,and the expression of key molecules of NF-κB signaling pathway was detected by Western blots.Whether TNFAIP3 affects the expression of inflammatory molecules in NAFLD cell model through NF-κB signaling pathway was verified by the agonist(LPS)or inhibitor(Aconine)recovery experiments of NF-κB signaling pathwayResults:1.The secretion of TNF-α,IL-6 and IL-1β were significantly up-regulated,and the expression of TNFAIP3 was significantly down-regulated in NAFLD cell model;2.Si-TNFAIP3 significantly promoted the expression of inflammatory factors in NAFLD cell model.Overexpression of TNFAIP3 can inhibit the expression of these inflammatory factors;3.Knock-down TNFAIP3 significantly increased the phosphorylation levels of ASK1,IκBɑ and p65 in NAFLD cell model,while TNFAIP3 overexpression caused an opposite effect.LPS treatement reversed the inflammatory response caused by TNFAIP3 overexpression,while Aconine reversed the pro-inflammatory response caused by TNFAIP3 silence..Conclusion: TNFAIP3 can inhibit the occurrence and disease development of NAFLD by negatively regulating NF-κB signaling pathway and its mediated inflammatory response,or inhibiting the activation of ASK1.Part Three miR-125 b regulates inflammatory responses in NAFLD cell mo-dels mediated by NF-κB signaling pathway by targeting TNFAIP3Objective: To explore the regulation of inflammatory response by miR-125 b and TNFAIP3 during the development of NAFLD based on cell model.Methods: Hep G2 cells were treated with free fatty acid(FFA)to construct cell model of NAFLD.The interaction between miR-125 b and TNFAIP3 was confirmed by dual luciferase reporter assay.The effects of expression of miR-125 b regulated by knockdown or overexpression of TNFAIP3 by cell transfection technique was observed.Expression of TNFAIP3 and p65 protein in Hep G2 cells was examined by immunofluorescence.Results:1.The expression of miR-125 b was significantly upregulated in an in vitro cell model of NAFLD;2.Downregulation of miR-125 b expression can significantly reduce TNF-α,IL-6 and IL-1β,while miR-125 b overexpression played the opposite role.3.TNFAIP3 is the direct downstream target gene of miR-125 b,and miR-125 b could negatively regulate the expression of TNFAIP34.miR-125 b inhibitor can reduce the phosphorylation levels of ASK1,IκBɑ and p65,but knockdown of TNFAIP3 can reverse this effect;miR-125 b mimic can enhance the phosphorylation levels of ASK1,IκBɑ and p65,but TNFAIP3 overexpression can reverse the effect.Conclusion: miR-125 b can activate NF-κB signaling pathway and exacerbate the inflammatory response in NAFLD by targeting TNFAIP3.Part Four miR-125 b regulates NF-κB signaling pathway-mediated infl-ammatory response in mouse model of NAFLD by targeting TNFAIP3Objective: To verify the inflammatory response mediated by miR-125 b regulates NF-κB signaling pathway by targeting TNFAIP3 in NAFLD mouse model.Methods: NAFLD mouse model was constructed by feeding mice with high fat diet.The expression of FFA and various inflammatory factors in mouse serum was detected by down-regulating miR-125 b and silencing TNFAIP3 by lentiviral transfection technology.HE staining was performed on liver tissues to detect the liver pathology of mice.Results:1.The level of FFA in the blood of the NAFLD mouse model was increased,the expression of miR-125 b in the liver was significantly increased,and the expression of TNFAIP3 was significantly decreased.;2.Knock-down miR-125 b had no significant effect on the level of FFA;however,further knock-down TNFAIP3 significantly increased the level of FFA;3.In miR-125 b inhibitor group,the weight,liver weight,the expression of inflammatory factors decreased significantly.However,further knockdown of TNFAIP3 can alleviate these effects.Conclusion: miR-125 b can affect the inflammatory response of NAFLD by targeting TNFAIP3.Conclusions:1.TNFAIP3 is a direct downstream target gene of miRNA125 b,which negatively regulates the expression of TNFAIP3.2.miRNA125 b can activate the NF-κB signaling pathway and aggravate the inflammatory response in NAFLD by directly targeting TNFAIP3.