| ObjectiveTo observe the effect of estrogen on modulating apolipoprotein AI gene transcription and investigate the role of different fractions of apo AI gene promoter and apoCⅢ/AⅣintergenic region in the transcription,to analyze the modulating mechanism of estrogen in apo AI gene expression.Methods1.Exposure HepG2 cells to different concentration of estradiol(0.01,0.1,1,10,20μmol/L) for 24 hours,respectively;HepG2 cells were incubated with estradiol at 10μmol/L concentration for 1,6,24,48 hour incubation.Then cells were collected for the isolation of RNA and the levels of mRNA were analysized by RT-PCR.The apo AI mRNA levels were expressed as the ratio of apo AI to P-actin.2.To construct recombinants containing different length DNA fragments of apo AI gene(-41/+397,-256/+397,and -2500/+397) with or without a 7-kb region apo CⅢ/AⅣintergenic region.Then these recombinants were cloned to a luciferase reporter plasmid.The final recombinants with the internal control vector pRL-null with renilla luciferase reporter gene were transfected to HepG2 cells by the cationic lipid method.After 24h,the cells were treated with estradiol at 10μmol/L concentration;After 48h,cells were collected and lysized.The activities of these luciferase enzymes were measured with Dual-GloTM Luciferase Assay System from Promega.Results1.HepG2 cells incubated with different concentrations of estradiol for 24 hours showed in a dose-dependent increase in the expression of apoAI mRNA.When estradiol concentration was 10μmol/L,the expression of apoAI mRNA was higher than that of 0,0.01,0.1,1μmol/L.Although the expression declined when estradiol was 20μmol/L,it still higher than 0,0.01,0.1,1μmol/L.2.A time-course experiment at the 10μmol/L of estradiol indicated that the expression of apoAI mRNA has no increased at 1 hour and significantly increased at 6 honx(P＜0.05).The expression was highest at 24 hour and then significantly declined at 48 hour.3.The luciferase activities of recombinant plasmids containing apoAI -256/+397bp and -2500/+397 DNA area was significantly increased than that only containing basal promoter(-41/+397bp) DNA fragment.The recombinants with or without apo CⅢ/AⅣintergenic region showed similar results.4.The recombinants activities of related luciferase enzymes with apo CⅢ/AⅣintergenic region were significantly increased than only containing the different fractions of apo AI gene promoter.5.In estradiol induced experiments,exception of the recombinant plasmids containing only the basal promoter(-41/+397bp) with or without region of apo CⅢ/AⅣ,The all recombinants expression of luciferase enzymes was great increased. There was no different between pGL2/-41A I and pGL2/-41A I CⅢAⅣin the presence of estradiol.Conclusion1.Estrogen may modulate apoAI gene transcription and show dose-dependent and time-dependent.The highest expression for apoAI was observed for estradiol at l0μmol/L concentration and the level of apoAI mRNA start to increased at 6h and significantly increased at 24h.2.The apo AI promoter area from the upstream of transcription site(-256bp -41bp)show higher transcription activities.It may be caused by this region containing the apo AI hepatic enhancer.Maybe there was no DNA element that can enhance the expression of apo AI in the region -2500- -256bp.There may contain some elements to regulate the expression of apo AI in apo CⅢ/AⅣintergenic region.3.Transcription activity was not affected by estrogen in the promoter area (-41/+397bp).Promoter area containing -256bp can increase transcription activity after stimulating by estrogen.It is inferred that this area may be the smallest reactive element for estrogen.The apo CⅢ/AⅣgene modulating was not affected by...
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