Farnesoid X Receptor Down-regulates Apolipoprotein M Expression Of Mechanism

Background Atherosclerosis (atherosclerosis, AS) is a commoncardiovascular and cerebrovascular diseases that has serious threat tohuman health, and has a higher incidence in the population of the East andthe West. Abnormal lipid metabolism is the important reason, But itsmechanism not yet completely expounded at present. Farnesoid x receptor(farnesoid x receptor, FXR) as a multi-functional transcription factor canregulate the expression of many target genes,and it is one of the membersof the nuclear receptor superfamily; FXR is highly expressed in liver, smallintestine and adrenal cortex.Which is involved in the regulation of bile acidmetabolism, lipid metabolism, glucose metabolism and cholesterolmetabolism.Apolipoprotein M is a newly discovered apolipoprotein which belongsto the lipocalin superfamily members, And it is highly expressed in liver, inkidney tube epithelial cell. Apolipoprotein M is extremely rich in content inHDL. which participates in the process of preβ-HDL and the reversetransport process of cholesterol, by which apoM plays inportant role inregulatory function.Our previous studies have shown: FXR was possible to decline the apoM expression in HepG2and in LO2cell. inhibition of FXR activity, theapoM expression increased. This proves that the apoM is very likely thatthe new target gene of FXR.To further expounded that FXR down-regulatesexpression of apoM that have the function of development in AS, toprevent and control AS that provides a new treatment way.Objective To Explore the effect of FXR down-regulates expressionof apolipoprotein M and the study of its mechanism.Methods (1) Through bioinformatics methods, online analysis theapoM gene promoter region, found FXR binding sequence, initiallydetermined apoM and FXR binding sites are located in the promoter regionwide. By chromatin immunoprecipitation（Chromatin immunoprecipitation,ChIP）analyze the interaction between FXR and apoM promoter regionsequence.(2)To construct the vector of apoM-siRNA,and directly transfectHepG2and L02cell, the expression of apoM mRNA was tested by realtime PCR in HepG2and L02cell; the expression of apoM protein wastested by Western Blotting in HepG2and L02cell.. The protein levelchange of the expression of apoA-I was tested by ELISA.Results (1) Analysis the apoM gene promoter region-3000bpsequence, to get FXR binding sites DR2, our previous studies have shownamplified apoM promoter region (-1900~-1800), the region contains thepredicted DR2; FXR agonist CDCA treated the HepG2cells, the group treated with anti-FXR antibody amplified177bp contains apoM promoterregion (-1900~-1800) at-1863bp by chip experiments, while the negativecontrol group is not significantly expanded.(2) After treated with directly transfection of apoM-siRNA for24-48hours in HepG2and L02cell, apoM mRNA and protein expression leveldecreased significantly compared with control (P<0.05);After After treatedwith Guggulsterone (GS)(10μM) for16-18hours in HepG2cell; GStreatment group: apoA-I protein expression level increased significantlycompared with control(P<0.05); apoM-siRNA-(+) transfeced with GSgroup: apoA-I protein expression level decreased significantly comparedwith GS treatment group (P<0.05).Conclusion (1) In HepG2cells, human apoM promoter sequencescontaining DR2(-1900~-1800) may combine with FXR.FXR and DR2(-1900~-1800) may combine to regulate transcription function.(2) we successfully constructed the vector of apoM-siRNA,GSpromoted the expression of apoA-I protein in the HepG2cell,which is to berelated with apoM.