| Objective: By comparing the effects of genistein(GEN)on the regulation of lipid metabolism in Hep G2 hepatocytes with those of 17β-estradiol(E2),this study aimed to explore the molecular mechanism of GEN-mediated regulation of lipid metabolism correlated with estrogen receptor β(ERβ),modulated micro RNA(mi RNA)and its target genes.It is expected to provide a new theoretical basis for the application of GEN in the prevention and treatment of lipid metabolism disorders in postmenopausal women.Methods: A model of Hep G2 cells eliminated environmental estrogenic influences was established by using phenol red-free DMEM medium and 10% charcoal/dextran-stripped fetal bovine serum.MTT assay was used to determine the intervention dose of GEN by detecting effects of GEN on the survival rate of Hep G2 cells.Hep G2 cells were divided into five groups: control(CON),E2 of 1 nmol/L(E2),GEN of 1μmol/L,10 μmol/L,25 μmol/L(G1,G10,G25).The cells were treated with both E2 and GEN for 24 h.TRNAzol lysates extracted of cells of CON and GEN group were collected and used for further mi RNA microarrays.Gene Ontology(GO)was used to analyze the function of differentially expressed mi RNA.PHTPP was treated with cells and used as an ERβ inhibitor.Cell transfection was used to up-regulate the level of mi R-363-3p and to down-regulate the level of phosphatase and tensin homologue deleted on chromosome 10(PTEN).In cell transfection experiments,Hep G2 cells were either divided into three groups as negative control(NC),mi R-363-3p mimic,mi R-363-3p mimic with GEN intervention,or NC,si-PTEN,si-PTEN with GEN intervention.GPO-PAP assay was used to detect the content of triglyceride(TG).Western Blot and RT-PCR were used to detect expression levels of proteins and genes related to lipid synthesis such as sterol regulatory element binding protein-1c(SREBP-1c),fatty acid synthase(FASN),stearoyl-Co A desaturase-1(SCD1);fatty acid β oxidation such as carnitine palmityl transferase 1α(CPT1α),perixisome proliferation-activated receptor α(PPARα);lipid transfer such as microsomal triglyceride transfer protein(MTTP).Additionally,expression levels of key regulators of lipid metabolism,kinase B(PKB or AKT)and mammalian target of rapamycin complex 1(m TORC1),were also detected.Moreover,expressions of ERβ、mi R-363-3p and PTEN which might be also modulated by GEN were evaluated in this study.Results:(1)Similar to the effects induced by E2,GEN also reduced the cellular TG content(P<0.05).In addition to that,GEN significantly reduced the protein and gene levels of lipid synthesis-related factors such as SREBP-1c,FASN and SCD1,increased the gene levels of factors related to fatty acid-oxidation and lipid transport such as PPARα,CPT1α and MTTP(P<0.05).Moreover,GEN inhibited phosphorylation levels of AKT and m TOR,which are key regulators of lipid metabolism(P<0.05);(2)After a pretreatment with the ERβ inhibitor,PHTPP,the inhibitory effect on modulating expressions of lipid synthesis,fatty acid-oxidation and lipid transfer by GEN were abolished(P<0.05).Similarly,the phosphorylation levels of AKT and m TOR inhibited by GEN were restored with pretreatment of PHTPP(P<0.05);(3)GEN down-regulated the expression level of mi R-363-3p and up-regulated the expression level of PTEN(P<0.05).PTEN could bind to the 3’-UTR of mi R-363-3p;(4)GEN abolished the increase of lipid synthesis due to the over expression of mi R-363-3p(P<0.05).The expression of mi R-363-3p was increased even with GEN intervention in Hep G2 cells with pretreatment of PHTPP(P<0.05);(5)GEN inhibited the lipid accumulation due to PTEN inhibition(P<0.05).PHTPP eliminated the up-regulation of PTEN expression induced by GEN(P<0.05).Conclusions:(1)GEN might reduce hepatic lipid synthesis and promote fatty acid β-oxidation through regulating AKT/m TOR signaling via ERβ;(2)GEN might exert a regulatory role in hepatocyte lipid metabolism through ERβ,which is associated with the down-regulation of mi R-363-3p and the promotion of the expression of its target gene Pten after GEN intervention. |
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