Epigenetic Study On LPK Gene In Nonalcoholic Fatty Liver

Non-alcoholic fatty liver disease (NAFLD) is threatening to become a major public-health. L-type pyruvate kinase (LPK) is a rate-limiting glycolytic enzyme, which can catalysis enolphosphopyruvate to pyruvic acid. The significant association between NAFLD and LPK gene has been consistently proved by few association studies. In the present study, we attempted to investigate epigenetic modifications of LPK gene in NAFLD model.Firstly, palmitic acid (PA)-induced NAFLD BRL cell model and HFD-induced NAFLD rat model were created. mRNA level and enzyme activity of LPK was dramatically reduced (P<0.01) in both model. Furthermore, significant reduction of LPK protein level in NAFLD BRL cell model was observed. Overexpression of LPK gene in human liver cell (QSG7701) could accelerates the cell proliferation (P<0.01), glucose consumption (P<0.01) and triglyceride transferation (P<0.05). Meanwhile, downregulation of LPK gene expression by siRNA can greatly reduce the cell proliferation, glucose consumption and triglyceride metabolism (P<0.01). These results supported that LPK can regulate the glucose and lipid metabolism. In addition, Breberine (BBR) significantly decreased mRNA level and enzyme activity of LPK in both model (P<0.01).Based on above results, epigenetic modifications (DNA methylation, histone acetylation and miRNA) of LPK gene in NAFLD model were investigated:1. The mean methylation levels of LPK promoter were increased in the both NAFLD model (P<0.05). Luciferase Reporter Assay showed that the elevated methylation level of LPK promoter lead to lower transcriptive activity (P<0.01). Furthermore, the elevated level of genomic DNA methylation by HPLC analysis (P<0.01) and decreased mRNA level of DNA methyltransferases gene (Dnmt1, Dnmt3a and Dnmt3b) by real-time PCR (P<0.05) were observed in both NAFLD models. Moreover, treatment of rat liver BRL cell with BBR could lead to decrease methylation of LPK promoter (P<0.05), partially increase mRNA level of DNA methyltransferases gene and genomic DNA methylation level(P<0.05).2. The acetylation level of histone H3and H4that around LPK DNA area was dramatically decreased in NAFLD model (P<0.01). BBR treatment led to lower acetylation level of histone H3and H4(P<0.05). The amount of ChREBP that binding to LPK promoter was notably decreased (P<0.01).3. A miR-15b combining site was predicted in the LPK3’-UTR region by software. The mRNA level of miR-15b was markedly elevated (P<0.01) in both NAFLD models. Overexpression miR-15b in QSG7701cell could accelerates the cell proliferation (P<0.01), glucose consumption (P<0.05) and triglyceride metabolism (P<0.05). The combining capacity between miR-15b and LPK3’-UTR was identified using Luciferase Reporter Assay, which was accompanied by significantly weaker reporter expression level. Our data also showed a significant reduction of miR-15b expression in serum of64fatty liver disease patients when compared to normal control (P<0.01). Serum miR-15b might serve as potential new biomarkers for NAFLD.In summary, the present study has demonstrated regulation mechanisms of LPK gene expression from epigenetic modifications, i.e., DNA methylation, histone acetylation and miRNA. Our findings also illustrate that BBR represents a promising agent for the treatment of NAFLD might through reversing epigenetic modifications and induce LPK gene expression.