β分泌酶-1基因甲基化与阿尔茨海默病的相关性研究  

Association between theβ-amyloid cleaving enzyme 1gene methylation and the genetic sensitivity of sporadic Alzheimer’s disease

作　　者：孙蓉[1] 肖琳[1] 宋明洁 何静杰[1] 贾建平 SUN Rong;XIAO Lin;SONG Ming-jie(China Rehabilitation Research Center,Beijing 100068,China)

机构地区：[1]中国康复研究中心神经康复K2科,北京100068

出　　处：《中国实验诊断学》2022年第4期475-480,共6页Chinese Journal of Laboratory Diagnosis

基　　金：国家重点基础研究发展计划(973计划)青年项目(2013CB837300)。

摘　　要：目的 探讨中国北方汉族人群β分泌酶-1(β-amyloid cleaving enzyme,BACE1)基因启动子区甲基化程度与散发性阿尔茨海默病(sporadic Alzheimer’s disease,SAD)发病的关系及机制。方法分析BACE1基因启动子区CpG岛,随机选取35名SAD患者、33名轻度认知障碍(mild cognitive impairment,MCI)患者和22名正常人,进行BACE1基因启动子区甲基化程度的测定,比较各组甲基化差异;构建报告基因质粒,转染细胞,测定荧火虫荧光素酶的活性(LAF)和海肾荧光素酶的活性(LAR),相对荧光素酶活性(RLA,RLA=LAF/LAR)来表示启动子质粒的转录活性。分别用血清剥夺、Aβ25-35、S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)及5-脱氧杂氮胞苷(5-aza-2′-deoxycytidine,5-aza-CdR)处理,检测不同处理后质粒的荧光素酶活性。结果 BACE1(-407至+60)启动子区有23个甲基化位点。其中大多数CpG位点甲基化程度很低,平均甲基化率小于10%。基因启动子区平均甲基化率与年龄存在负性线性相关。Aβ25-35干预增加SY5Y细胞中BACE1启动子质粒的转录活性约20%;SAM干预明显降低BACE1启动子质粒的转录活性。血清剥夺及5-aza-CdR干预下的RLA与非处理组相比均无显著性差异(P>0.05)。结论 BACE1基因甲基化程度的改变与SAD发病有明显相关性。SAM可以通过诱导甲基化抑制基因的转录活性,从而影响淀粉样蛋白分泌酶的活性,进一步导致AD发病。Objective To study the association between theβ-amyloid cleaving enzyme 1(BACE1)gene methylation and the genetic sensitivity of sporadic Alzheimer’s disease(SAD).Methods The CpG islands in the promoter of BACE1gene were analyzed and predicted by Methyl Primer Express software.Masscleave method was used to perform an analysis of DNA methylation in the promoters of BACE1gene in lymphocytes of 35SAD patients、33mild cognitive impair(MCI)patients and 22age-and gender-matched controls.We used the genetic recombination methods to build reporter gene plasmids,then transient transfected into the SH-SY5Yand HeLa cells.Firefly luciferase activities(LAF)and renilla luciferase activities(LAR)were measured sequentially using a Dual-Luciferase reporter assay system.The relative luciferase activity(RLA)was calculated as:RLA = LAF/LAR.To explore the role of environmental risk factors on transcriptional activities of different promoters,we treated cells with several agents after they were transfected,including serum deprivation(to produce energy starvation and apoptosis),Aβ25-35(to mimic the model of AD),5-azaCdR(to inhibit DNA methyl transferase)and SAM(to induce transmethylation).Results We found 23potential methylation sites in the region-407to+60of the BACE1promoter.The majority of CpG sites were methylated to a very low degree,with average methylation below 10%.Some CpG sites of BACE1(Cytosines at-167,-46,-19,+23and+28)showed hypomethylation patterns in SAD cases compared with MCI and control groups.The frequencies of methylcytosines decreased with age in the analyzed regions.Reporter assay showed that under Aβ25-35treatment,the BACE1transcriptional activity was 20%increase compared with control only in SH-SY5Ycells(SH-SY5YP=0.000;HeLa P=0.092).We also found significant difference under SAM treatment in both cell lines(P<0.01).Serum deprivation and 5-aza-CdR treatment had no significant effect on transcriptional acitivities of BACE1gene promoters(P>0.05).Conclusion The hypomethylation of CpG islands in the promoter of

关 键 词：阿尔茨海默病 认知障碍 启动子 甲基化 Β分泌酶 

分 类 号：R741.05[医药卫生—神经病学与精神病学]