The Study Of Induction On Silenced Fragile X Mental Retardation-1 Gene Expression By Adenylate Cyclase Activator

Fragile X syndrome is the most commom form of heritable mental retardation. The disease account for X-linked mental reatadation is about 40%, and account for non-specificial mental reatadation is about 2%～6%. Fra (X) is associated with an unstable expansion of the CGG trinucleotide repeats in 5' untranslating region and abnormal methylation of the CpG island in the promter region of the fragile X mental retardation-1 gene (FMR1). As the transcription is blocked, fragile X mental retardation protein (FMRP) expression is absence. The distribution of FMR1 is extensive, including brain,testis and peripheral blood mononuclear cells (PBMC). Generally speaking, methylation is more important than expansion of trinucleotide in pathogenic mechanism.Confirmation had showned that there was a methylation sensitive element (MSE) in the FMR1 promter, and there was a cAMP-responsive element (CRE) in MSE. Studies had showned that the cAMP level reduced in the lymphocytes and platelets of Fra (X) patients and in cells model in vitro. Besides that, there was negative correlation between the size of expansion of trinucleotide in FMR1 and intra-cellular cAMP. And the increasing of cAMP was along with the increasing of the expression of FMRP. All datas showed that there could be regulation between FMR1 and cAMP. Up to now, there is no investigation about elevating cellular cAMP by drugs would reactivate silenced FMR1.Previous study on two key enzymes in metabolic process of cAMP in silenced FMR1 cells model, the result had indicated that the inhibition on activity of adenylate cyclase (AC) may be the main reason of reduced cAMP in cells from patients affected Fra (X). In this experiment, firstly, we tried to rasie cAMP by drugs to reactivate silenced FMR1, and then compared induction of three drugs, a special activator of adenylate cyclase,a classical demethylated drug and a histone acetylase inhibitor, on silenced FMR1 mRNA expression, aiming to explore the possibility of drugs which could rasie cellular cAMP on reactivation silenced FMR1, and provide a new method and a new clue on therapeutics.Methods:1,Applied sodium nitroprusside (SNP) into medium to silence FMR1: Sodium nitroprusside can activate DNA methylatransferases abnormally, and block up the combinding of nucleate factors with promter, resulting in inhibiting of FMR1 mRNA transcription. PC 12 cells are originated from phaeochromocytoma of rat, and they are served as nerver cells models to study in nerve physiology,nerve biology and neuropharmacology generally. Sodium nitroprusside can silenced FMR1 in peripheral blood mononuclear cells and PC12 cells succeefully. The differences in two cell models were concentrations of the sodium nitroprusside, in other words, the concentration of sodium nitroprusside silenced FMR1 in peripheral blood mononuclear cells was 500umol/L, while in PC12 cells was 1000umol/L. In these Fra (X) cell models, sodium nitroprusside should be changed within 48 hours to keep FMR1 silenced. Observed silenced FMR1 by reverse transcriptase PCR (RT-PCR).2,Forskolin, a special activator of adenylate cyclase to reactivate silenced FMR1:12 hours after FMR1 silenced by sodium nitroprusside, added forskolin to cell medium in Fra(X) cell models of peripheral blood mononuclear cells and PC 12 cells respectively. And then collected cells aftrer forskolin affecting cells 24 hours, extracted RNA, tanned RT-PCR, and observed FMR1 mRNA expression.3,The comparison of induction by three drugs on silenced FMR1 in peripheral blood mononuclear cells: 12 hours after silenced FMR1 by sodium nitroprusside, forskolin, 5-Aza-dC and sodium butyrate were added to medium respectively. And then collected cells, extracterd RNA, runned semi-quantitative of RT-PCR and compared the changes of FMR1 mRNA expression under Doc Gel 2000 scanning imaging system after drugs affecting cells 12 hours,24 hours,36 hours,48 hours and 72 hours. And numerus were analysised through statistics software SPSS 11.5.4,The comparison of induction by three drugs on silenced FMRI in PC 12 cells: Be similar with peripheral blood mononuclear cells modle, the differences in observation moments were every 24 hours.5,The testing of gene sequences in blank control and in reactivated silenced FMR1 gene by forskolin through Chromas software and BLAST software.Results:1,The influences of sodium nitroprusside on FMR1 in peripheral blood mononuclear cells and PC12 cells: There were two bands in blank control lane, one stands for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) about 250bp, and the other for FMR1 about 500bp, and sequences from blank control were coincidenced with Genebank report. In two cell models of Fra (X), sodium nitroprusside can silence FMR1 successfully in 12 hours,24 hours and 48 hours, as there was one band about 250bp appearing at that moment. But there were two bands in 72 hours lane, implyed that present of FMR1 transcription. If changed medium with flesh sodium nitroprusside within 48 hours, the influences of sodium nitroprusside on silenced FMR1 would be persistent. Besides that, the morphouses of PC12 cells did not change obviously with or without sodium nitroprusside under microscope.2,The influences of forskolin on silenced FMR1 in peripheral blood mononuclear cells: Forskolin could reacivate silenced FMR1 after affecting 24 hours, and sequences from reactivated FMR1 by forskolin were coincidenced with Genebank report. It was assumed that forskolin would induced silenced FMR1 expression in vitro.3,The influences of forskolin on silenced FMR1 in PC12 cells: Forskolin could reacivate silenced FMR1 after affecting 24 hours. It was assumed that forskolin would induced silenced FMR1 expression in vitro.4,The comparison of induction by three drugs on silenced FMR1 in peripheral blood mononuclear cells: Forskolin could induced silenced FMR1 expression in 24 hours,36 hours,48 hours and 72 hours respectively. And 5-Aza-dC could also induced silenced FMR1 expression in 12 hours,24 hours,36 hours,48 hours and 72 hours respectively. These datas indicated that effect by forskolin was later than 5-Aza- dC's. The peak values of action by 5-Aza-dC and forskolin were at 48 hours, and account for transcription level of blank control was 75.59% in forskolin. The degression of forskolin was gentler and induction tendency was longer than 5-Aza-dC's. Besides that, there were significant differences in forskolin group and 5-Aza-dC group (p＜0.05). While sodium butyrate could not induced silenced FMR1 effectivelly.5,The comparison of induction by three drugs on silenced FMR1 in PC 12 cells: Forskolin and 5-Aza-dC could induced silenced FMR1, and the induction characteristics,action peak time and tendency of two drugs were similar with peripheral blood mononuclear cells models. But there was no significant differences in forskolin group and 5-Aza-dC group (p＞0.05). The peak values of induction by forskolin account for transcription level of blank control was 76.83%. And there was no significant differences in inductions in two cell models by forskolin. While sodium butyrate could not induced silenced FMR1 effectivelly.Conclusions:1,Learned of silencing FMR1 in peripheral blood mononuclear cells by sodium nitroprusside, it could silence FMR1 in PC 12 cells successfully with concentration of 1000umol/L. It's the first time to construct Fra (X) model with PC12 cells. And it would be very important to investigate Fra(X) under the situation lacking animal models with this simple and reliable cell model.2,Rasing cellular cAMP by applying adenylate cyclase activator to study the possibility of induction in aboved-mentioned Fra(X) cell models. And results suggested that forskolin could reactivate silenced FMR1, and this reactivation was about 75% compared with blank control transcription. It was the first time to find that rasing cAMP could induce silenced FMR1 expression, and this data indicated that there would be correlation between FMR1 and cAMR And it would be a new clue in therapy of Fra (X).3,Compared with forskolin, an activator of adenylate cyclase and 5-Aza-dC, a classical demethylation drug, both of two drugs could reactivate silenced FMR1 effectively and their action peak values were identical. But effect of forskolin was later and gentler than 5-Aza-dC's. And tendency of forskolin was longer than 5-Aza-dC's. While sodium butyrate could not induced silenced FMR1.