The Study Of The Mechanism Of Induction On Silenced Fragile X Mental Retardation-1Gene Re-expression By Adenylat Cyclase Activator

Fragile X syndrome[Fra(X)] is the most common inherited mental retardation.At present, the pathogenesis mechanismt of Fra (X) is mainly thought that the the CGG three nucleotide repetitive sequences in fragile X mental retardation-1gne(FMR1) promotor increases unstablely and the upstream CpG island happen unusual methylation, and these sudden changes cause the FMR1gene duplication suppressed, so cause its code product fragile X mental retardation protein to reduce or to lack, finally Fra (X) performed. At present it believed that whether the CpG island happened unusual methylation is more improtant than the number of CGG three nucleotide repetitive sequences for the influence to phenotypes.Many previous studies to prompt that mental retardation and signal transduction pathway has some contact with each other, and the cAMP signaling pathway is most closely related to learning,memory and intellectual, so the relationship between the Fra (X) and the cAMP pathway caused people close attention. It was found that, in the cells of patients with Fra (X), in the brain tissue and platelets of FMR1gene knockout mice, as well as suffering from Fra (X) of the Drosophila brain tissue,cAMP levels were lower than the normal. And those suffering from fra (X) of Drosophila can compensate for this loss by re-implantation of the FMR1gene, which imply the mutually regulation relationship between the FMR1gene and the cAMP.Why cAMP levels in the cells of Fra (X) patients decreased? cAMP metabolism is realated with adenylate cyclase (AC) and phosphodiesterase (PDE) activity. The former make the ATP generate cAMP, whereas the latter degradation of cAMP to maintain the balance of the intracellular cAMP level. Clinical study found that part of the Fra (X) of patients with the dyfunction of the catalytic subunit or regulation adenylate cyclase.In earlier period we has studied activity of two key enzymes, namely:adenylate cyclase(AC) and phosphodiesterase(PDE) after the FMRl gene been silenced by the methylation. And discovered the cAMP level drops in the cells,and the activity of adenylate cyclase reduces obviously, but activity of phosphodiesterase(PDE) is not remarkable different after FMR1gene been silenced.From this, we proposed the functional defects of the FMR1gene can affect adenylate cyclase activity, suggesting that the adenylate cyclase activity may affect intracellular cAMP levels in the patients with Fra (X).If the FMR1gene transcription and translation termination is related with the low activity of adenylate cyclase, on the contrary, can we use drugs increase the activity of adenylate cyclase to restart the FMR1gene has been closed?In order to prove our conjecture,our preliminary studies found that adenylate cyclase activating agent-forskolin could induce silenced FMRl gene re-expression on the cell closed, but we are not sure that forskolin induced silenced FMR1gene re-expression whether by increasing the cAMP levels to achieve?Has confirmed that the transcription of the FMR1gene and promoter region of the FMR1gene is closely related.Has confirmed that the FMRl gene promoter region having methylation-sensitive areas (MSE), and the methylation-sensitive areas contains the sequence of a cAMP response unit (CRE). MSE and CRE’s structural are overlap. CRE sequence is cyclic adenosine monophosphate response element binding protein (CREB) binding sites. CREB is an important nuclear transcription factor, regulating a wide range of biological functions, including memory function. And found that CREB can bind the FMR1promoter and may enhance the activity of the FMR1factor. Therefore we speculated that the mechanism may be the cAMP induced. Whether the AC agonist-forskolin through the cAMP pathway, through the the FMR1promoter region of the MSE/CRE overlap to regulate the FMR1gene has not to be confirmed.To further confirm the above speculations,1. This study attempts to use new clinical cAMP drugs-two of dibutyryl cyclic adenosine monophosphate (db-cAMP)to induced silenced FMR1gene transcription and protein expression, further confirmed one of the ways of the adenylate cyclase activator re-expression is by increasing the intracellular cAMP levels;2.Denylate cyclase activator if through the sequence of methylation sensitive areas of MSE/CRE overlapping?Also plan to take MSE/CRE overlapping sequence in FMR1gene promotor, changing CRE sequence to formate the mutant promoter M1, M2respectively,constructing Dual-Luciferase Reporter System to further analyze the relationship between activity of the CRE sequence and activity of promotor methylation, to confirm forskolin regulate of the FMR1gene through FMR1promoter region of the MSE/CRE overlapping sites and lay the foundation for discussing the mechanism of adenylate cyclase activating agent make FMR1gene re-express.Research technique:1. Make pc12cell model of FMR1gene silenced:Make model using sodium nitroprusside (SNP), nitric oxide(NO) donor, providing NO which activate the DNA methyltransferase (DNA MeTase), making cytosine of MSE sequence in CpG island of FMR1gene methylate, thus block the nucleic acid factors link the promotor, and suppress the FMR1mRNA duplication. Observing the FMR1gene silenced effect through the real-time PCR.2.Observe the effect of db-cAMP on the FMR1gene in the pc12mRNA level: After culturing24h with SNP1000umol/L adding db-cAMP (finally density0.25mmol/L,0.5mmol/L,1mmol/L) continue cuiture and collect cell separately in6h,12h,24h,48h for got total RNA and compare with normal group(Npc12) or inhibition group that pc12cells only plus SNP1000umol/L. Real-time PCR (dye method) to detect the expression of the FMR1gene.3. Observe the effect of db-cAMP on FMRP expression:After raising24h with SNP100umol/L adding db-cAMP0.5mmol/L and continues to raise24h,48h,72h,96h(every12hours adds db-cAMP and every48hours adds SNP)),finally collect cells again, observing situation of FMRP expression.4. Construct fluorescein enzyme carrier:Get the MSE/CRE overlapping sequence from K562cells total DNA by the way of PCR. And then using the PCR point mutation method to mute the MSE sequence to form a mutant M1, M2respectively. Last,the MSE, Ml, M2fragments were connected to the PGL3vector respectively,finally transfect into DH5cells. Extract plasmid MSE/PGL3, M1/PGL3, M2/PGL3respectively with the Renilla plasmid co-transfection in pc12cells, then measuring luciferase enzyme activity.The MSE/PGL3, M1/PGL3, M2/PGL3group compare the negative control group respectively (the pGL3Basic+Renilla plasmid),the difference was statistically significant, which means plasmid successfully transfected into pc12cells and the MSE/CRE overlapping sequence for studing of the luciferase reporter gene system was successfully constructed.5. Verify that the CRE is the key point of the adenylate cyclase activator-forskolin induced silenced FMR1gene expression: Extracting MSE/PGL3, M1/PGL3, M2/PGL3plasmid from the DH5bacterial and transfecting into pc12, latter continues to culture.Next,adding SNP1000mmol/LAND(OR) forskolin50umol/L in different groups.Last, Detect luciferase activity and compare the effect of forskolin in normal CRE and M1,M2. By comparison of the value of luciferase to the infer of the promoter activity (the luciferase value is high means the promoter activity is high; on the contrary, it is lower).By detecting of promoter activity to verify the CRE sites is adenylatecyclase activating agent-forskolin to restart the silenced FMR1gene locus.6. Statistics analysis:All data were analyzed using the SPSS13.0statistics software, All data were expressed as mean(X)±standard deviation (SD). The fluorescence quantitation PCR result’s is used2-ΔΔCT value to analyze, and data use the completely randomized designed One-Way ANOVA during group, and the multiple comparisons use the Dunnett T3test because it don’t conform to the normal distributio;The Westernblot data is used ratio which is a value by analyzing with the Bia-Rad software, statistical analysis were performed by the completely randomized designed One-Way ANOVA during group,and the multiple comparisons with LSD test;The data of relative fluorescein enzyme activity is used relative fluorescein enzyme activity intensity value to analyze, statistical analysis were performed by Factorial design ANOVA and the multiple comparisons with LSD test; The above all significance levels were set at P<0.05for all statistical analyses.Results:1.The effects of db-cAMP on silenced FMR1transcription in different concentration and different time poin:db-cAMP can rstart FMR1gene in level of mRNA at db-cAMPO.5mmol/L12h (P<0.005)after SNP silenced FMR1gene. The db-cAMP0.5mmol/L12h play strongest effect on mRNA activation. The quantity increase to0.86times of the normal group(Npc12group,P=0.135), and is9.7times of the inhibition group (P<0.05). The maintain effect of db-cAMP0.5mmol/L is less than18hours.2.The effect of db-cAMP on expression of FMRP:Results show that SNP can decrease FMRP expression. FMRP expression was the lowest at72h, It is quantity approximately0.56times as the normal group. In the SNP induces FMR1silenced group, adding0.5mmol/L db-cAMP can enhance FMRP expression,and the best activation effect at72h.It is quantity approximately0.98times as normal group(P<0.05), is approximately1.25times as silenced group (P<0.05).3. Construct fluorescein enzyme carrier:PCR product, double enzyme validation product were sequencd and the sequencing results are in accord with design.After pGL-3/MSE, pGL-3/Ml,pGL-3/M2plasmid and Renilla plasmids are co-transfected into pc12cells respectively, the luciferase activity is measured and the results are analyzed compareing with the negative control group. There are statistics significance, indicating plasmid successfully transfected into pc12cells. And the Dual-Luciferase Reporter gene system aiming at MSE/CRE overlapping sequence is successfully constructed.4. Verify that the CRE is the key point of the adenylate cyclase activator-forskolin inducing silenced FMR1gene expression:(1) PGL-3/MSE, pGL-3/M1or pGL-3/M2plasmid and Renilla plasmid co-transfected pc12cells and then treated only by SNP. compared with the control group,MSE group, M1group or M2group were statistically significant (P<0.05),implying the FMR1have been closed successfully by SNP treatment;(2) PGL-3/MSE, pGL-3/M1or pGL-3/M2plasmid and Renilla plasmid co-transfected into pc12cells and then treated only by FSK. there is on significant difference compare MSE group, M1group, M2group with the control group (P> 0.05), implying when the CRE sequence structure is normal or mutant, forskolin play no open role in the normal FMR1gene;(3) PGL-3/MSE, pGL-3/M1or pGL-3/M2plasmid and Renilla plasmid co-transfected pc12cells. After the treatment of SNP adding FSK, the promoter active in MSE group was significantly higher than group only added SNP,there is no significant difference between SNP+FSK group and only SNP control group, when mutate MSE/CRE sequences forming M1and M2,there is on difference between the SNP+FSK group and only SNP control group. Implying under the CRE structure does not change the circumstances, forskolin can open the closed FMR1gene,while change the CRE sequence, the opening function does not exist.Conclusions:1. Confirmed new cyclic adenosine monophosphate (db-cAMP) can be effective to restart silenced FMR1gene and make protein expression. This discovery confirmed that increasing the intracellular cAMP levels is one of the mechanisms of inducing silenced FMR1gene re-expression,providing a new thought and a new method of therapy for Fra (X) patients in gene level.2. Verify that one of the cAMP activator-forskolin open the FMR1closed gene is achieved by increasing the intracellular cAMP levels.3.Construction pGL-3/MSE, pGL-3/M1, pGL-3/M2plasmid reporter system successful. According to the results,when the CRE loci in normal, FSK can open FMR1promoter effectively; when the CRE mutated, ther opening effect is significantly reduced on FMR1promoter.This result confirmed the CRE loci is the key sites of FSK re-open FMR1promoter, which suggesting forskolin open FMR1gene may be mediated by the promoter region of the MSE/CRE overlapping sequence.