The Study Of Transcriptional Regulation On Apo AⅠ Gene By Estrogen And Gemfibrozil

Objection: To investigate Apolipoprotein A I (ApoA I )gene expression and the transcription effiency by the estrogen and gemfibrozil.To determine the potential mechanisms responsible for the estrogen and gemfibrozil responsiveness of the regulatory element of ApoA I promoter.Methods:HepG2 cells were incubated with l,10,20,30μmol/L estrogen or 20,40,50,60ug/ml gemfibrozil for 24hours.Then cells were collected for the isolation of RNA and nuclear proteins . HepG2 cells were incubated with 10μmol/L estrogen or 40ug/ml gemfibrozil for 1,6 and 24hours. Then cells were collected for the isolation of RNA and nuclear proteins at 1,6 and 24hours. We measured ApoA I mRNA expression by RT-PCR.Using electrophorese mobility shift assays(EMSA) ,we measured the interactions of the nuclear proteins from HepG2 cells exposure to estrogen with synthetic labeled A probe, we also measured the interactions of the nuclear proteins from HepG2 cells exposure to gemfibrozil with synthetic labeled DRE probe .Results:(1)Incubation of HepG2 cells with different concentrations of estrogen for 24 hours resulted in a dose-dependent increase in the expression of ApoA I mRNA. When estrogen was10, 20 and 30μmol/L, the expression of apoA I mRNA was higher than 1μmol/L.A time-course experiment showed the expression of ApoA I mRNA was not increased at 1hour when treated with 10μmol/L estrogen ,slightly increased at 6hours, and significantly increased at 24hours.(2)EMSA reaction showed A probe can bind with the nuclear proteins from HepG2 cells treated with estrogen .When estrogen was 1μmol/L ,the amounts of nuclear proteins which were binded to the A probe was not changed. The more estrogen were treated ,the more nuclear proteins were binded to the Aprobe.The amounts of nuclear proteins which were binded to the A probe was higher at 24hours than at lhour. The competition mobility shift experiment showed that 50-fold molar excess of unlabeled A probe can partly repress the binding of labeled A probe and nuclear proteins ,while 100 and 200-fold molar excess of unlabeled A probe can completely repress the binding of labeled A probe and nuclear proteins .However no competition was observed with the equvalent molar excess of unlabeled B and ERE probe.(3) Incubation of HepG2 cells with different concentrations of gemfibrozil for 24 hours resulted in a dose-dependent increase in the expression of ApoA I mRNA. When gemfibrozil was40, 50 and 60ng/ml, the expression of ApoA I mRNA was higher than 20ug/ml. A time-course experiment indicated that when cells were treated for 1,6 and 24hours in 40p.g/ml gemfibrozil ,the expression of ApoA I mRNA was gradually increased when the time goes by. (4)EMSA reaction showed DRE probe can bind with the nuclear proteins from HepG2 cells treated with gemfibrozil. When gemfibrozil was 20ug/ml,the amounts of nuclear proteins which were binded to the DRE probe was not changed. The more gemfibrozil were treated ,the more nuclear proteins were binded to the DRE probe. When treated with 40ug/ml gemfibrozil, the amounts of nuclear proteins was gradually decreased when the time goes by. The competition mobility shift experiment showed that 100-fold molar excess of unlabeled DRE probe can partly repress the binding of labeled DRE probe and nuclear proteins ,while 200 and 400-fold molar excess of unlabeled DRE probe can completely repress the binding of labeled DRE probe and nuclear proteins .However no competition was observed with the equvalent molar excess of unlabeled API and AP2 probe.Conclusion: (DApoA I gene expression is regulated at the transcription level by estrogen.lt increased the expression of apoA I mRNA by estrogen dependent...