纳米金电化学信号放大技术应用于铝暴露认知功能障碍相关micro RNAs分析

Alzheimer’s disease (AD) is a regressive disease of central nervous system (CNS).The dysregulation of miRNA expression has been found to be closely associated with AD.Our previous work showed high aluminum exposure may be a risk factor of AD in the mining area.The data also suggest that about 17 related AD locus might not be associated with high aluminum exposure in both Zhuang or Han population. A small sample of individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays show 7 miRNAs displayed significantly different expression levels in high aluminum exposure serum compared with controls. Amplified voltammetric detection of miRNA sequences in the large sample was carried out by nanotechnology. A biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode.Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with Fc-capped gold nanoparticle /streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10fM and 2.0 pM. The low detection levels allow the direct quantification of miRNA to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for the detection of nucleic acids in clinical samples.

老年性痴呆（AD）是一种神经退行性疾病，与遗传和环境因素关系密切。MicroRNAs(miRNAs) 直接或间接参与与AD发病相关基因表达调控。我们前期工作表明，高铝暴露是广西矿区内AD发病的危险因素，AD相关基因17个位点基因型与铝暴露致认知障碍无统计关联。用定量-逆转录实时荧光聚合酶链反应技术检测小样本发现，7种miRNAs在铝暴露认知障碍组和对照组存在显著差异，而大样本量高昂代价使得高通量测序难以维系。纳米技术在生物医学应用，新型高灵敏高准确低代价检测miRNAs方法日趋成熟。项目通过biotin-miRNA与待测miRNAs竞争与固定在芯片表面的DNA序列杂交，并引入二茂铁覆盖纳米金-亲和素复合物放大电化学信号，检测大样本铝暴露认知障碍相关miRNAs含量，应用统计软件分析环境高铝暴露认知障碍组与对照组miRNAs之间差异表达，探寻潜在的AD早期诊断标记物及其简单低价高效检测方案。

累计完成铝矿区4874例老年人认知能力调查，铝矿区高铝暴露老年人群10年前检测约为13.16%，现为16.85%，明显上升，不容乐观，亟需积极应对。小样本共计106例发现7种miRNAs在铝暴露认知障碍组和对照组存在显著差异，7种miRNAs为：hsa-miR-103a-3p，hsa-miR-107， hsa-miR-26b-5p，hsa-miR-98，hsa-miR-342-3p，hsa-let-7d-3p，hsa-miR-125a-5p，样本量增加后有4种miRNAs在铝暴露认知障碍组和对照组存在显著差异，4种miRNAs为：hsa-miR-103a-3p、hsa-miR-107、hsa-miR-26b-5p、hsa-miR-98，提示随样本量增加铝暴露认知障碍组和对照组miRNAs表达趋于复杂，因不能明确铝暴露认知障碍敏感miRNAs。高通量测序检测提示铝暴露正常与健康对照相比，miRNA的预测靶基因有11396个，铝暴露认知障碍与健康对照相比，miRNA预测靶基因有10435个，铝暴露认知障碍与铝暴露正常相比预测靶基因有1335个。与健康对照相比，铝暴露认知障碍、铝暴露正常组均以下调为主。利用KEGG数据库对预测得到的靶基因进行Pathway分析发现，铝暴露认知障碍、铝暴露正常与健康对照三者相比差异miRNA所调控的靶基因通路在MAPK信号通路均有富集。