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

Research background:The fragileⅩsyndrome{Fra (Ⅹ)}is one of most common hereditary mental retardation diseases. According to the conservative estimate, our country has 200,000 Fra (Ⅹ) patients at least. The patients have normal life, but the intelligence is low and does not have the independent life ability, therefore they create the enormous burden to the family and the society.At present, the pathogenesis mechanismt of Fra (Ⅹ) is mainly thought that the the CGG three nucleotide repetitive sequences in fragileⅩmental retardation-1 gne(FMR1) promotor increases unstablely and the upstream CpG island happen unusual methylation, and these sudden changes cause the FMR1 gene duplication suppress, so cause its code product fragileⅩmental retardation protein to reduce or to lack, finally Fra (Ⅹ) perform. 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. Pietrobono R discovery that witnout DNA's methylation, the CGG repetitive sequence itself don't prevent the translation of FMR1,as well as the FMRP product normally. Had the case to report that the patient whose (CGG) n of FMR1 gene has increased to changes completely, but as a result of the CpG island don't happening methylation, FMRP still can be able to express normally, the fragileⅩmental retardation protein is also normal. So it consider that whether there is methylation perhaps is primary cause for the FMR1 gene duplication silence and the corresponding protein expression lack. Reduction or absence of FMRP cause the fragileⅩsyndrome. FMRP is a RNA binding protein,which plays an important regulatory role in shearing,RNA transport,mRNA stability of transcribing level and translation level through the target RNA.Many studies suggest that FMRP can affect formation and function of synapse by regulating mRNA translation and protein synthesis. In dendritic spine FMRP affect brain development and synaptic plasticity by regulating of protein synthesis,. In the synapse, if the lack of FMRP, some proteins are over-translation, resulting in enhancement of long-term depression (LTP) which relate to learning and memory,and ultimately lead to mental retardation and behavioral problems. Besides for diagnosis testing express of FMRP can better reflect the level of mental retardation of patients than detecting DNA. Overall, the study of FMRP is very important for pathogenesis, clinical diagnosis and treatment of Fra (Ⅹ). Hwu and so on researchers discoveries that there is a methylation sensitive area (MSE) in promotor of the FMR1 gene which can have enhancer activity and relates with duplication of FMRl gene. This MSE sequense and cAMP responded unit (CRE) overlaps, but the CRE sequence can link with phosphoric acid adenosine response unit protein (CREB). CREB is one kind of important nucleus transcription factors, can adjust widespread biology function including the memory function, and is one kind of universal existence adjustment members which is necessery for forming of the long-term memory. Other researchs discover the level of cAMP in the cell drops equally for Fra (Ⅹ) patient as well as in vitro model FMR1 gene silence, the number of (CGG)n three nucleotide fragments in FMR1 gene increase with the level of cAMP dropping,and the output cAMP in cell increases along with the FMR1 code protein-fragileⅩmental retardation protein's expression. The above material prompt that the between FMR1 gene and the level of cAMP possibly exists mutually regulating relations.In earlier period we has studied activity of two key enzymes, namely:adenylate cyclase(AC) and phosphodiesterase(PDE) after the FMR1 gene silence by the methylatingin cAMP metabolism process, and discovered after FMR1 gene silence the cAMP level drops in the cell,and activity of adenylate cyclase reduces obviously, but activity of phosphodiesterase(PDE) is not remarkable different. From this, we proposed that the lose of FMR1 gene's function may affect the activity of adenylate cyclase, and the activity of adenylate cyclase suppressing possibly one of primary resences which causing the cAMP's level to reduce in the Fra (Ⅹ) patient's cell. This research attempts to use adenylate cyclase activating agent forskolin(FSK) to enhance the adenylate cyclase activity, improves the low cAMP level position in the FMR1 gene cell, discusses the effect on silencing the FMR1 gene's duplication and the protein expression by the medicine inducing,and plan to take MSE/CRE overlapping sequence in FMR1 gene promotor in the way of PCR, construct Dual-Luciferase Reporter System to further analyze the relationship between activity of the CRE sequence and activity of promotor methelation, to lay the foundation for discusses the mechanism of adenylate cyclase activating agent make FMR1 gene reexpress, thus provides the new treatment and thought of Fra(Ⅹ).Research technique:1.Cell culture:Culture K-562 and the Hela cell useing the RPMI-1640 complete nutrient fluid and 10% newborn cow blood serum; the cell generat in 37℃and in 5% CO2 saturated humidity's incubator. Replace culture medium interval 2 to 3 days. Continue to culture evading the light after adding SNP.2. Make K-562 cell model of FMR1 gene silence:Make model using sodium nitroprusside (SNP), nitric oxide(NO) the donor, provide NO which activate the DNA methyl transferase (DNA MeTase), makes cytosine of MSE sequence in CpG island of FMR1 gene methylate, thus blocke the link the nucleic acid factors wich the promotor, and suppress the FMR1 mRNA duplication.2.1 Select density of SNP:Appling ordinary and Taqman fluorescence quantitation PCR observes the FMR1 gene silencing effect, according to the literature divides into the normal group (to add double steams the water (SNP to use this thing configuration)), the SNP500umol/L group, the SNP800umol/L group, the SNP1000umol/L group (to join separately corresponding finally density SNP), according to result select best density.2.2 Observe the effect of SNP on the FMR1 gene in the mRNA level:Appling two kind of PCR observe the FMR1 gene silencing effect, supposes separately 5 time point (12h,24h,48h,72h,96h,96h) (72h replaces SNP), observes duration after joining disposablely the SNP, and whether having continually silencing effect after replaceing SNP.2.3 Observe the effect of SNP on the FMRP expression:Using the Western blot method, takes separately 3 time piont (24h,48h,72h), observes situation of FMRP the expression.3. Observation the effect of adenylate cyclase activating agent (FSK) on FMR1 gene in mRNA level:Useing SNP1000umol/L, and after culturing 24h add FSK (finally density 50μmol/L) continue cuiture and collect cell separately in 12h,24h,48h, the 72h, and supposes normal group (here adds DMSO (FSK to apply this thing configuration)), after applying the above two PCR method observes restarting effect the to FMR1 gene. 4.Observe the effect of FSK on FMRP expression after restarting the FMR1 silencing gene:With western the blot method, after raising 24h with SNP(1000umol/L) adds FSK (density 50μmol/L) and continues to raise 72h,finally the collect cell again, observes situation of FMRP expression.5. Construct fluorescein enzyme carrier:5.1 Take MSE/CRE overlap senquence in promotor of FMR1 gene:Extract genome team DNA according to takara material particle extraction reagent box instruction booklet convention increases, take fragment with PCR,latter connect the PCR product with the T carrier, and withdraws plasmid T-MSE, finally confirmate the sequence by senquencing.5.2 Constructthe expression vector:T-MSE as the template, obtains fragment which have kpnⅠand hindⅢendonuelease sites using the PCR, afterward uses restrictive interior contact enzyme kpnⅠand hindⅢtogether with the PGL4 carrier plasmid to proform enzyme digestion, confirmat using PCR, the double enzyme cuting and the sequencing, finally connect to construct the expression vector.6.Exam activity of the Dual-Luciferase Reporter System6.1 Cell tranfection:Lay hela cell in logarithm vegetal period into 24-well plate by 105 each hole's density,make mixture according to the Lipofectamine2000 Transfection reagent manual (24-well each quantity):PGL-4/MSE fluorescein enzyme report plasmid or PGL-4.Basic 5ul plus Renilla plasmid 0.5ul plus lipo2000 3.5ul, and altogether transfection, latter continues to culture.6.2 Exam relative fluorescein enzyme activity:Collects the above cell,and determinat fluorescein enzyme activity refer to Dual-Luciferase Reporter the Assay reagent box explanationThe above experiments are repeatde 3 times in the same condition,finally average value. 7. Statistics analysis:All data were analyzed using the SPSS 13.0 statistics software, All data were expressed as mean(Ⅹ)±standard deviation (SD),except data of FMRP express using the average order time (M) because it don't conform to the normal distribution; The fluorescence quantitation PCR result's is used 2-ΔΔCT value to analyze, except data SNP time point statistical analysis were performed by the completely randomized designed Kruskal-Wallis test during group,and the multiple comparisons with Bonferroni test,all other data use the completely randomized designed One-Way ANOVA during group, the multiple comparisons use the SLD test; 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 Kruskal-Wallis test during group,and the multiple comparisons with Bonferroni test; The data of relative fluorescein enzyme activity is used relative fluorescein enzyme activity intensity value to analyze, statistical analysis were performed by two independent samples T-tests; The above all Significance levels were set at P<0.05 for all statistical analyses.Results:1. Make K-562 cell model of FMR1 gene silence successfully1.1 For SNP of 500,800 and 1000μmol/L concentration, there is a certain sealing effect (F=105.423,P<0.001).After dealing with, the levels of FMR1 gene expression were reduced to 0.063,0.017 and 0.006 times of control group, and the effect of SNP of 1000μmol/L is the most obvious (P<0.001), lasts for 96H after adding SNP one time, and can continue to if changed when culture 72h.1.2 The effect of SNP on the FMRP expression:At 24h,48h and 72h after SNP silencing FMR1 gene, FMRP expression's quantity decrease to 0.807,0.697,0.477 times of the control group. It's show that SNP can inhibit significantly expression of FMRP afte scliencing FMR1 gene. (F= 149.117,P<0.001)2. The rstarting effect of FSK on the FMR1 gene in level of mRNA: FSK can rstart FMR1 gene in level of mRNA at 24H (F=34.921, P<0.001; P<0.001) after SNP silencing FMR1 gene, the quentity increase to 0.133 times of the control group, and can continue to 48H(F=34.921, P<0.001; P<0.001), then its quantity is 0.138 times of the control group.3. The effect of FSK on expression of FMRP:Result show that FSK can inhibite significantly the expression of FMRP (P=0.57), the quantity of FMRP increase to one times of the control group.4.Contruction of luciferase vector:All the sequences in steps (MSE sequence, the MSE sequence after adding restriction sites) sequence rightly, the vector was constructed successfully,and be identified by PCR, restriction enzyme digestion methods,result of sequencing is also successful.5. Exam relative fluorescein enzyme activity of Dual-Luciferase Reporter gene system:After pGL-3/MSE plasmid and Renilla plasmids are transfected hela cells, the luciferase activity is measured and the results are analyzed.Compareing with the negative control group, there are statistics significance, indicating plasmid successfully transfected hela cells. And the Dual-Luciferase Reporter gene system aiming at MSE/CRE overlapping sequence is successfully constructed. Conclusions:1. Make K-562 cell model of FMR1 gene silence successfully,and discover the quantity of FMR1 gene's expression decrease down to 0.006 times after SNP dealing with, and quantity of FMRP fall to 0.477 times of the control group, So the methods to make Fra (Ⅹ) cell model is feasible.2.FSK can increase the activity of adenylyl cyclase, improve the low level of cAMP of the FMR1 gene's cell,and may restart FMR1 gene in the mRNA level aftear silencing by methylating,and the quantity of the gene expression increase up to 0.138 times of the control group, continue to 48 hours, but the qutantity of FMRP expression is close to the normal group after 72 hours.This result show adenylyl cyclase activator drugs is feasible for the treatment of Fra (Ⅹ),and can provide new ideas and therapeutic prospects for the the treatment of Fra (Ⅹ).3. Our experiment tranfer successful the MSE/CRE overlapping sequence of FMR1 gene promoter region by PCR method, and successfully construct MSE/CRE overlapping sequence/PGL-4 dual luciferase reporter gene system,and lay a basis for further analyzing the relationship between activity of CRE sequence activing by adenylate activation agent and methylation of FMR1 gene promoter and studing the mechanism of the drug induceing silencing FMR1 gene transcript and express again.