腺苷酸环化酶激活剂诱导脆性X智力低下一号基因重新表达的机制研究

发布时间：2018-06-11 14:23

  本文选题：脆性X智力低下一号基因(FMR1) + 双荧光素酶报告基因系统　； 参考：《南方医科大学》2012年硕士论文

【摘要】：脆性X综合征[fragile X syndrome,Fra(X)]是最常见的遗传性智力低下疾病之一。据保守估计,我国至少有20万脆性X病人。目前认为Fra(X)致病机制主要是由于脆性X智力低下一号基因(fragile X mental retardation-1gene,FMR1)启动子区内(CGG)n三核苷酸重复序列的不稳定扩增及其上游的CPG岛的异常甲基化,使FMR1基因转录及翻译终止,其编码产物-脆性X智力低下蛋白(fragile X mental retardation protein, FMRP)减少或缺如,最终表现为Fra(X)。目前对于Fra(X)的治疗尚无有效的方法,基因治疗仍困难重重,药物重新活化FMR1的方法,也因其存在较强的毒副作用制约了临床应用,寻找有效、安全、低/无毒副作用的药物用于治疗Fra (X)仍是目前研究的热点之一 已往很多研究提示智力低下与信号转导通路之间有着某种相互联系,其中,环磷酸腺苷(cyclic adenosine monophosphate,cAMP)信号通路与学习记忆和智力关系最为密切,故Fra(X)与cAMP通路之间的关系也引起了人们的密切关注。人们发现在Fra(X)患者细胞中、在FMR1基因敲除小鼠的脑组织及血小板内,以及在患有Fra(X)的果蝇脑组织内cAMP水平均低于正常,而患有Fra(X)的果蝇可以通过重新植入FMR1基因来弥补这种缺陷,由此提示FMR1基因与cAMP之间可能存在相互调控关系。 Fra(X)患者细胞内cAMP水平降低的原因是什么呢?cAMP的代谢主要与腺苷酸环化酶(adenylate cyclase,AC)和磷酸二酯酶(phosphodiesterase,PDE)的活性有关。前者作用于ATP可生成cAMP,而后者则降解cAMP,以保持胞内cAMP水平的平衡。有临床研究发现一部分Fra(X)患者存在腺苷酸环化酶催化亚单位的功能或者调节的异常。我们前期研究发现FMR1基因封闭后细胞内cAMP水平下降,腺苷酸环化酶活性也明显降低,而磷酸二酯酶的活性则无显著变化。提示FMR1基因的功能缺陷可影响腺苷酸环化酶的活性,推测腺苷酸环化酶活性抑制可能是影响Fra(X)患者细胞内cAMP水平降低的主要原因。 如果FMR1基因转录及翻译终止与腺苷酸环化酶活性降低有关,反之,用药物提高腺苷酸环化酶的活性能否重启被封闭的FMR1基因呢?为了证明我们的猜想,前期在封闭的FMR1基因细胞模型上,我们采用特异性腺苷酸环化酶(AC)激活剂药物弗司可林(forskolin,FSK)改善腺苷酸环化酶的低活性状态,结果发现它的确可有效地重新诱导沉默FMR1基因的转录和蛋白表达,但forskolin对FMR1基因调节的具体机制尚不清楚,是否通过提高cAMP水平这一途径诱导的有待进一步证实。 已知FMRI基因启动子区与FMR1基因的转录密切相关,该区内存在一个具有增强子活性的甲基化敏感区(1methylation sensitive element,MSE),值得注意的是该区增强子内又同时包含一个cAMP反应单位(cAMP-responsive element,CRE)碱基序列,两者结构重叠,关系紧密。而CRE序列是环磷酸腺苷反应单元结合蛋白(CREB)的结合位点。CREB是一种重要的核转录因子,具有调节包括记忆功能在内的广泛的生物学功能,且研究发现CREB可以与FMR1启动子结合并可增强FMR1启动子的活性,因此推测其机制可能是cAMP诱导的。AC激动剂forskolin是否经cAMP通路,通过FMR1启动子区内的MSE/CRE重叠位点实现对FMR1基因调节的尚有待证实。 为了证实以上推测,本研究1.拟直接采用外源性cAMP药物-二丁酰环磷酸腺苷(db-cAMP)诱导沉默FMR1基因的转录和蛋白表达,试图证实forskolin是通过提高细胞内cAMP水平这一途径而达到对FMR1基因再激活作用的；2.以MSE/CRE位点为研究靶点,构建FMR1启动子区MSE/CRE位点双荧光素酶报告基因系统,在不影响MSE整体启动子活性的情况下,分别突变CRE序列,形成突变启动子M1、M2的双荧光素酶报告基因,试图验证forskolin是经FMR1启动子区MSE/CRE位点实现了对FMR1基因的调节,为进一步研究AC激动剂forskolin重启FMR1基因的机制奠定基础,试图为Fra(X)患者的治疗提供新的思路和治疗前景。 研究方法： 1.制作pc12细胞FMR1基因封闭的细胞模型 采用硝普钠(sodium nitroprusside,SNP)封闭FMR1基因。硝普钠为经典的一氧化氮(nitric oxide,NO)供体,可通过产生的NO活化DNA甲基转移酶(DNA MeTase),从而使FMR1启动子区CpG岛的MSE中的胞嘧啶发生高度甲基化,阻断了核酸因子与启动了的结合,抑制了FMRl mRNA的转录。最后通过real-time PCR观察FMR1基因的封闭效果。 2.观察二丁酰环磷酸腺苷(db-cAMP)对pc12细胞FMRl mRNA的影响 1000umol/LSNP作用pc12细胞24h后,实验组分别加入浓度为0.25mmo1/L0.5mmo1/L、1mmol/L的db-cAMP,分别于db-cAMP作用6h、12h、24h、48h后收集细胞,提取总RNA。以正常的pc12细胞为正常对照组,以仅加SNP作用的pc12细胞为封闭抑制对照组,用荧光定量PCR(染料法)检测FMR1基因的表达情况。 3.观察二丁酰环磷酸腺苷(db-cAMP)对FMR1蛋白水平(FMRP)的影响 硝普钠封闭pc12细胞FMR124h后,加入0.5mmol/Ldb-cAMP,分别于加药24h、48h、72h、96h后收集细胞(12小时补充失效的db-cAMP,48小时补充失效的SNP),提取蛋白质,进行Western Blot,对FMRP进行半定量分析。 4.FMR1启动子MSE/CER重叠序列及突变CRE双荧光素酶报告基因的构建 采用PCR的方法从K562细胞DNA中钓取MSE/CRE重叠序列,然后采用PCR点突变的方法分别突变CRE序列,形成突变了M1、M2,分别获得MSE、M1、M2片段,然后连接到PGL3载体上,进而转化到DH5a细胞中,提取质粒。MSE/PGL3、M1/PGL3、M2/PGL3分别与海肾质粒共转染pc12细胞后,对所测荧光素酶活性强度值进行分析。 5.验证CRE位点是腺苷酸环化酶激活剂forskolin重启FMR1基因的关键位点 从DH50细菌中1提取MSE/PGL3、M1/PGL3、M2/PGL3质粒,分别转染pc12细胞后进行分组,加1000umol/L SNP和(或)50umo1/Lforskolin,检测荧光素酶活性,比较在正常CRE与突变后CRE状态下,forskolin对FMRl基因开启的影响,通过对荧光素酶值的比较,判断启动子活性(荧光素酶值高,启动子活性则高；反之,则低),以此验证CRE位点是否是腺苷酸环化酶激活剂forskolin重启FMR1基因的关键位点。 6.统计 实验所得数据经SPSS13.0统计软件进行统计学处理,数据用均数士标准差(χ-±s)表示。荧光定量PCR结果采用2-△△cT值分析,采用单因素方差分析；因方差不齐,组间两两比较采用Dunnett T3;与正常组的比较采用单样本t检验。WesternBlot数据采用Bia-Rad Gel-Pro Analyzer软件分析所得A值之比,因方差齐性,组间两两比较采用LSD；相对荧光素酶活性检测数据应用相对荧光素酶活性强度值分析,采用析因设计资料方差分析,因方差齐性,组间两两比较采用LSD。p0.05表示有统计学差异。 研究结果 1.不同浓度及不同时间点db-cAMP对封闭的FMR1转录水平的影响 db-cAMP在0.5mmo1/L作用12h时FMR1mRNA激活效果最强,FMR1mRNA的表达量约为封闭组的9.7倍(P0.05),约为正常组的0.86倍(P=0.135)。0.5mmol/Ldb-cAMP的激活效果维持时间小于18小时。 2.db-cAMP对FMRP表达的影响 用SNP1000μmol/L封闭FMR1基因,作用72h时FMRP表达量最低,约为正常组的0.56倍；在SNP封闭的基础上,加入0.5mmol/Ldb-cAMP可提高FMRP表达量,作用72h时激活效果最好,为正常组的0.98倍(P0.05),是封闭组的1.25倍(P0.05)。 3.荧光素酶载体构建 PCR、双酶切验证后送测序,测序结果均符合设计。pGL-3/MSE位、pGL-3/M1位、pGL-3/M2位质粒、pGL3basic与海肾质粒共转染pc12细胞,质粒均成功转入pc12细胞,并且具有启动子活性,显示脆性X智力低下一号基因启动子双荧光素酶报告基因载体构建成功。 4、腺苷酸环化酶激活剂forskolin重启FMR1基因CRE位点验证 (1)经SNP封闭作用后,MSE位、M1位、M2位三组与未加SNP的各自正常对照组比较均有统计学意义(P0.05),提示经SNP作用,三组FMR1均被SNP封闭成功。 (2)未经SNP封闭,单经forskolin处理后,MSE位、M1位、M2位三组分别与各自的对照组相比较均无统计学意义(P0.05),提示forskolin对正常活性的FMR1启动子,无论CRE序列结构正常或突变,均无增强作用。 (3)已被SNP封闭后的FMR1,再添加forskolin处理,当CRE位点正常时,MSE组的荧光素酶活性显著高于未添加forskolin的封闭对照组(P0.05)；在突变了CRE结合位点的M1组和M2组中,添加forskolin组与未添加forskolin封闭对照组均无显著性差异(P0.05)。由此提示,当FMR1启动子活性低下时,在CRE结构正常情况下,forskolin可开启封闭的FMR1基因,而当突变CRE序列后,forskolin对FMR1封闭基因的开启作用则消失。 以上结果显示当CRE位点正常时,forskolin对活性低下的FMR1启动了有显著地激活作用,而对正常活性的FMR1启动子则无增强作用；当CRE位点突变之后,forskolin对正常活性、低下活性的FMR1启动子均无开启作用。 结论 1.发现直接采用外源性环磷酸腺苷(db-cAMP)也可有效地重新诱导被封闭的FMR1基因和蛋白表达,这有望为Fra(X)发病机制的研究和临床治疗提供新的思路和治疗前景。 2.证实了cAMP激活剂forskolin开启FMR1封闭基因的机制之一是通过提高细胞内cAMP水平而实现的。 3.成功构建FMR1启动子区MSE/CRE位点双荧光素酶报告基因系统,结果显示当CRE位点正常时,forskolin对活性低下的FMR1启动子有显著地激活作用,而当CRE位点突变之后,forskolin则无此作用,由此证实CRE位点是forskolin重启FMR1基因的关键位点。推测AC激动剂forskolin可能主要是通过FMR1启动子区内的MSE/CRE重叠位点,经cAMP通路实现了对FMR1基因的调控。
[Abstract]:Fra ( X ) is one of the most common hereditary mental retardation diseases . It is estimated that there are at least 200,000 fragile X patients in our country . At present , it is considered that Fra ( X ) pathogenesis is mainly due to the unstable amplification of ( CGG ) n trino - nucleotide repeat sequence in the promoter region ( CGG ) of fragile X mental retardation - 1 gene ( FMR1 ) and the abnormal methylation of CpG islands upstream .

There is a certain correlation between the mental retardation and the signal transduction pathways in many researches . The relationship between the signal pathway of cyclic adenosine monophosphate ( cAMP ) and learning and memory and intelligence is closely related . It is found that in Fra ( X ) patient cells , the level of cAMP in brain tissue and platelets of mice bearing Fra ( X ) is lower than that of normal , and that of Fra ( X ) can compensate for this deficiency by re - implanting the FMR1 gene , which suggests that there may be mutual regulation between FMR1 gene and cAMP .

In this study , we found that the activity of adenylyl cyclase ( cAMP ) decreased and the activity of adenylyl cyclase ( cAMP ) decreased and the activity of adenylyl cyclase ( cAMP ) decreased .

If FMR1 gene transcription and translation termination are related to the decrease of adenylyl cyclase activity , whether the activity of adenylyl cyclase can be restarted by drugs ? In order to prove our guess , we use the specific adenylyl cyclase ( AC ) activator , forskolin ( FSK ) , to improve the low activity state of adenylyl cyclase . The results show that it can effectively re - induce the transcription and protein expression of the silencing FMR1 gene , but the specific mechanism forskolin ' s regulation of FMR1 gene is not clear .

It is known that the promoter region of the FMRI gene is closely related to the transcription of FMR1 gene . In this region , there is a methylation sensitive element ( MSE ) with enhancer activity , which is a binding site of cAMP - responsive element ( CRE ) .

In order to confirm the above assumption , this study is intended to investigate the transcription and protein expression of FMR1 gene induced by exogenous cAMP - induced silence FMR1 gene . It is attempted to demonstrate that forskolin is activated by increasing cAMP level in cells .
2 . Using the MSE / CRE site as the research target , the MR1 promoter region MSE / CRE site dual luciferase reporter gene system was constructed . In the absence of the overall promoter activity of MSE , the CRE sequence was mutated respectively to form the dual luciferase reporter gene of the mutant promoters M1 and M2 . The attempt to verify that forskolin was the mechanism of the FMR1 promoter region MSE / CRE site was used to establish the basis for further study the mechanism of the AC agonist forskolin to restart the FMR1 gene , and to provide a new thought and treatment prospect for the treatment of Fra ( X ) patients .

Study method :

1 . Production of pc12 cell FMR1 gene - enclosed cell model

The FMR1 gene was blocked by sodium nitrogenase ( SNP ) . The classical nitric oxide ( NO ) donor could be activated by NO to activate DNA methyltransferase ( DNA MeTase ) , so that cytosine in the MSE of CpG island of FMR1 promoter was highly methylated , blocking the transcription of FMRl mRNA . Finally , the closing effect of FMR1 gene was observed by real - time PCR .

2 . To observe the effect of dibutyrylamide ( db - cAMP ) on the expression of FMRl mRNA in pc12 cells

After 24 h of pc12 cells , the cells were harvested at 6h , 12h , 24h and 48h at 6h , 12h , 24h and 48h respectively , and the total RNA was extracted . The normal pc12 cells were used as control groups , and the expression of FMR1 gene was detected by fluorescence quantitative PCR ( dye method ) .

3 . Observe the effect of dibutyrylamide ( db - cAMP ) on FMR1 protein level ( FMRP )

After blocking pc12 cell FMR124h , 0.5 mmol / L db - cAMP was added . After 24 h , 48 h , 72 h , 96 h after drug administration , the cells were collected ( 12 hours supplemented with failed db - cAMP , 48 hour supplemented failed SNP ) , protein was extracted , Western Blot was performed , and semi - quantitative analysis was performed on FMRP .

4 . Construction of the FMR1 promoter MSE / CER overlapping sequence and mutant CRE dual luciferase reporter gene

The MSE / CRE overlapping sequences were isolated from K562 cell DNA by PCR , and then the CRE sequences were mutated by PCR - point mutation . The MSE , M1 , M2 were amplified by PCR and then ligated to the PGL3 vector , and then transformed into the DH5a cells . Then , the plasmids were extracted . The MSE / PGL3 , M1 / PGL3 , M2 / PGL3 were co - transfected with the marine renal plasmid pc12 cells , and the intensity values of luciferase activity were analyzed .

5 . Verify that the CRE site is the key site of the Adenylate Cyclase activator forskolin to restart the FMR1 gene

The effect of forskolin on the opening of FMRl gene was determined from DH50 bacteria by extracting MSE / PGL3 , M1 / PGL3 , and M2 / PGL3 plasmids , respectively .
Conversely , low ) , thereby verifying whether the CRE site is a key site for the adenylyl cyclase activator forskolin to restart the FMR1 gene .

6 . Statistics

The data of the experiment was statistically processed by SPSS 13.0 , and the data was expressed by mean standard deviation ( 蠂 ~ 卤 s ) . The results of fluorescence quantitative PCR were analyzed by 2 - 鈻，

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