| Background: Nonalcoholic fatty liver disease(NAFLD)affects obesity-associated metabolic syndrome,which exhibits hepatic steatosis,insulin insensitivity and glucose intolerance.A high-fat diet(HFD)promotes the development of NAFLD and stimulates excessive accumulation of triglycerides and insulin resistance in the liver.NAFLD has become another major disease leading to end-stage liver disease after viral hepatitis,with its incidence increasing every year and no satisfactory treatment currently available.Emerging evidence suggests that micro RNAs(miRNAs)are essential for metabolic homeostasis of liver tissues.Many hepatic miRNAs located in the miR-379/miR-544 cluster were significantly increased in leptin-receptor-deficient type 2 mice(db/db),a mouse model of diabetes.Studies have shown that Dlk1(Delta-like homolog 1)and Igfl1(insulin-like growth factor 1 receptor)play important roles in regulating nutrient metabolism and insulin resistance,however,in obesity-induced NAFLD and other metabolic dysfunctions,the potential molecular connections between Dlk1,Igf1 r and miR-379/miR-544 clusters are mostly unclear and deserve further study,in order to identify the new function of miR-379/miR-544 clusters in the regulation of liver steatosis and metabolic syndrome and provide clues for this miRNA cluster to serve as a potential therapeutic target for obesity and NAFLD.Objective: To determine the role of the miR-379/miR-544 clusters in regulating hepatic steatosis and metabolic syndrome;To explore the potential molecular relationships between miR-379/miR-544 clusters and Dlk1,Igf1 r in the regulation of liver lipid metabolism,and provide clues for miR-379/miR-544 cluster as potential therapeutic targets for obesity and NAFLD.Methods: The miR-379/miR-544 cluster of systemic knockout mice(KO)were constructed,and mice were fed a chow diet with 10% kcal fat(Research Diet D12450B)or a high-fat diet with 60% kcal fat-containing diet(Research Diet D12492)for 14weeks(the knockout mice intervention model was abbreviated as KO-HFD,and the wild-type mice intervention model was abbreviated as WT-HFD).Glucose-tolerance tests were carried out on mice that had been fasted for 16 hours.The levels of triglyceride and cholesterol in livers,serum,and cultured cells were determined.The liver tissue,epididymal fat pad and cultured cells were collected and embedded in paraffin for H&E staining and oil red O staining.Extracting total RNA for RNA isolation and quantitative PCR analysis;Western blot was used to detect the protein levels of IGFIR,DLK1 and AKT phosphorylation(Ser 473)in the liver of WT-HFD and KO-HFD mice.HepG2 cells were cultured,transfected and treated with palmitic acid(PA).Detecting luciferase activity by luciferase report analysis;Bisulfite modification of genomic DNA extracted from liver of WT-HFD mice and KO-HFD mice using a bisulfite kit;GO and KEGG were used to analyze the differential expression of mRNAs in the liver.Results: 1.Expression levels of the miRNAs(miR-379,miR-411,and miR-543)located in the miR-379/miR-544 cluster were significantly increased in the liver of db/db mice and HFD-fed mice.2.The body weight gains of the miR-379/miR-544 KO-HFD mice were smaller than those of WT-HFD mice,*P<0.05,**P<0.01;Compared to WT-HFD mice,liver fat deposition was reduced in KO-HFD mice,epididymal adipocytes were significantly reduced,and liver triglycerides(TG),serum TG,serum glucose,and serum cholesterol levels were significantly reduced,*P<0.05.3.Compared with WT-HFD mice,the glucose tolerance and clearance of KO-HFD mice were significantly improved,*P<0.05;The mRNA levels of lipogenesis related genes(Pparγ,Fas and Scd1)in KO-HFD mice were decreased,*P<0.05.The expression levels of IGF1 R,DLK1,and AKT phosphorylation(Ser 473)in the liver of KO-HFD mice were significantly increased,**P<0.01.4.HepG2 cells were treated with 300μM palmitic acid(PA),a toxic lipid,and the expression level of miR-379 was increased three-folds,*P<0.05.When the miR-379 inhibitor was added,the expression of miR-379 in HepG2 cells was inhibited,and at the same time,lipid accumulation and cell triglyceride content were decreased in the presence of PA.Oil red O staining showed that the miR-379 mimic promoted lipid accumulation in HepG2 cells.In HepG2 cells overexpressed with miR-379,the cell triglyceride content was increased,*P<0.05.5.Analysis of relevant data reported by luciferase showed that Igf1 r and Dlk1 were direct target genes of miR-379/miR-544 cluster,*P<0.05.6.The expression of liver-related genes in KO-HFD mice is dysfunctional and the methylation status of DMR between genes is changed.Conclusion: Our results revealed a previously unknown function of the miR-379/miR-544 cluster in resistance to moderate hepatic steatosis,suggesting that the miR-379/miR-544 cluster might represent a potential therapeutic target for the treatment of NAFLD. |
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