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阿尔茨海默病(Alzheimer′s disease,AD)是一种表现为认知功能减退的神经退行性病变,其病程长、治疗费用高,给病人家庭和社会带来了沉重的负担[1]。然而,AD病因迄今未明,发病机制学说亦多[2]。寻找AD特异性标志物及研究其机制仍是对AD早发现、早诊疗的有效手段。microRNAs(miRNAs)是一类长为19~22 bp的内源性单链非编码小分子RNA[3],与包括AD在内的许多神经退行性疾病的发生及发展密切相关[4],因而也具有作为AD早期诊断的生物标志物的潜能。本研究从GEO数据库获取AD相关miRNAs芯片数据集,使用在线工具GEO2R分析差异表达的miRNAs,并选取明显高表达的miR-182-5p进一步验证。此外,本研究使用生物信息学对miR-182-5p进行系统的靶基因预测及功能分析,以期为深入研究其具体作用机制提供理论基础。
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GSE104514芯片数据来自12只AD模型小鼠(Tg)和12只野生型小鼠(wild)的全脑组织或大脑皮层。根据P < 0.05和| logFC |>1筛选获得6个差异表达miRNAs,其在小鼠的表达水平见图 1。其中,miR-182-5p在模型小鼠大脑组织中为差异倍数最大且唯一上调的miRNA(见表 1),因此选择miR-182-5p并对其进行深入分析。
基因名称 B LogFC t P miR-M1-2-3p -3.48 -2.01 3.10 < 0.01 miR-877* -3.74 -2.52 2.69 < 0.05 miR-182 -4.02 2.78 2.25 < 0.05 miR-10a -4.03 -1.20 2.23 < 0.05 miR-10b -4.03 -1.20 2.23 < 0.05 miR-17* -4.09 -1.84 2.13 < 0.05 表 1 miRNAs表达水平的差异
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应用qPCR检测AD组和对照组血清中miR-182-5p的表达水平,结果显示,AD病人血清中的miR-182-5p表达量为2.022±1.225, 高于对照组中miR-182-5p表达量0.486±0.442,差异有统计学意义(t′=5.95,P < 0.01)。
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通过miRBase数据库比较miR-182-5p在不同物种间的成熟体序列,结果显示miR-182-5p的序列在人(Homo sapiens)、家鸡(Gallus gallus)、豚鼠(Cavia porcellus)等10个物种中高度相似,表明miR-182-5p在不同物种间具有高度的保守性(见表 2)。
序列号 名称 序列 物种 MIMAT0038556 cli-miR-182-5p UUU GGC AAU GGU
AGA ACU CAC ACUColumba livia MIMAT0037544 gga-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACU GGallus gallus MIMAT0036764 oha-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACU GOphiophagus hannah MIMAT0037798 cpi-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUChrysemys picta MIMAT0047699 dno-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUDasypus novemcinctus MIMAT0048285 ocu-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUOryctolagus cuniculus MIMAT0047048 cpo-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUCavia porcellus MIMAT0038194 ami-miR-182-5p UUU GGC AAU GGU
AGA ACU CAC ACUAlligator mississippiensis MIMAT0038980 pbv-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUPython bivittatus MIMAT0000259 hsa-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUHomo sapiens 表 2 不同物种间miR-182-5p的序列比较
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运用miRcode、miRDB、miRWalk和Target Scan数据库预测miR-182-5p的下游靶基因,分别获得281、1 266、6 475、1 325个,结果取交集,共得到23个靶基因(见图 2),23个交集靶基因(见图 3)。
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首先,应用DAVID version 6.8(https://david.Ncifcrf.gov)数据库对miR-182-5p交集靶基因进行基因ID转换,随后将转换后的基因ID输入KOBAS version3.0 (http://kobas.cbi.pku.edu.cn/kobas3/)分析平台,对23个交集靶基因行GO和KEGG富集分析,以P < 0.05作为筛选界值。GO富集结果显示,miR-182-5p靶基因主要参与于锌离子的结合(zinc ion binding)、生物过程的负调控(negative regulation of biological process)、杂环类化合物的结合(heterocyclic compound binding)、主要代谢过程的调节(regulation of primary metabolic process)、gtpase的活性(GTPase binding)、膜的内在成分的组成(intrinsic component of membrane)、大脑的发育(brain development)、部分细胞形态的发生(cell part morphogenesis signaling)、转移酶的活性(transferase activity)、信号的调节(regulation of signaling)、泛素结合酶的结合(ubiquitin conjugating enzyme binding)、耳蜗的发育(cochlea development)、组蛋白h4-k16的乙酰化作用(histone H4-K16 acetylation)、咽系统的发育(pharyngeal system development)、神经元演化方向的规范(cerebral cortex cell migration)、大脑皮层细胞的迁移(cerebral cortex cell migration)、大脑皮层细胞的放射状定向迁移(cerebral cortex radially oriented cell migration)等多个生物学过程。前20个GO功能项见表 3与图 4。而KEGG通路富集分析结果显示,这些靶基因主要参与肾集合管的酸的分泌(collecting duct acid secretion),醚脂类的代谢(ether lipid metabolism),志贺菌病的致病过程(shigellosis),细胞黏膜的黏附(adherens junction),细菌对上皮细胞的侵袭(bacterial invasion of epithelial cells)等通路(见表 4、图 5)。
GO编号 名称 n P 基因 0008270 zinc ion binding 3 < 0.01 YAF2, ISL1, RBM5 0048519 negative regulation of biological process 4 < 0.01 YAF2, ISL1, RBM5, PAFAH1B1 1901363 heterocyclic compound binding 4 < 0.01 PHF20, ISL1, RBM5, TRIO 0080090 regulation of primary metabolic process 4 < 0.01 ANKIB1, ISL1, PHF20, MEOX1 0051020 gtpase binding 2 < 0.01 TBXA2R, TRIO 0031224 intrinsic component of membrane 4 < 0.01 SLC7A14, TSPAN9, ADAM22, SLC39A9 0007420 brain development 2 < 0.01 ISL1, PAFAH1B1 0032990 cell part morphogenesis 2 < 0.01 ISL1, PAFAH1B1 0023052 signaling 4 < 0.01 TRIO, SYPL1, TSPAN9, PAFAH1B1 0016740 transferase activity 3 < 0.01 PHF20, PDK4, TRIO 0023051 regulation of signaling 3 < 0.01 ISL1, PHF20, PAFAH1B1 0031624 ubiquitin conjugating enzyme binding 1 < 0.01 ANKIB1 0090102 cochlea development 1 < 0.01 PAFAH1B1 0043984 histone h4-k16 acetylation 1 < 0.01 PHF20 0060037 pharyngeal system development 1 < 0.01 ISL1 0048665 neuron fate specification 1 < 0.01 ISL1 0046972 histone acetyltransferase activity(h4-k16 specific) 1 < 0.01 PHF20 0003924 gtpase activity 2 < 0.01 TRIO, PAFAH1B1 0021795 cerebral cortex cell migration 1 < 0.01 PAFAH1B1 0021799 cerebral cortex radially oriented cell migration 1 < 0.01 PAFAH1B1 表 3 miR-182-5p靶基因GO分析结果
通路编号 名称 n P 基因编号 hsa04966 Collecting duct acid secretion 1 < 0.05 6521 hsa00565 Ether lipid metabolism 1 < 0.05 5048 hsa05131 Shigellosis 1 < 0.05 7414 hsa04520 Adherens junction 1 < 0.05 7414 hsa05100 Bacterial invasion of epithelial cells 1 < 0.05 7414 表 4 miR-182-5p靶基因KEGG分析结果
miR-182-5p在阿尔兹海默病的表达及生物信息学分析
Expression and bioinformatics analysis of miR-182-5p in Alzheimer's disease
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摘要:
目的探究miR-182-5p在阿尔兹海默病(AD)病人血清中的表达水平,并采用生物信息学方法对其进行靶基因预测及功能分析。 方法从GEO数据库获取AD相关miRNAs芯片数据集GSE104514,利用GEO2R在线工具分析差异表达的miRNAs,选择差异最大的miR-182-5p进行深入研究。收集AD病人28例(AD组)及健康者15名(对照组),应用实时荧光定量聚合酶链反应法检测血清中miR-182-5p的表达水平。通过miRBase数据库分析miR-182-5p在不同物种间的保守性;运用miRcode、miRDB、miRWalk和Target Scan数据库预测miR-182-5p靶基因,并取交集靶基因;应用DAVID和KOBAS数据库对miR-182-5p靶基因进行GO功能及KEGG信号通路富集分析。 结果AD小鼠脑组织的miR-182-5p表达明显高于野生型小鼠(P < 0.05),AD病人血清中的miR-182-5p表达量(2.022±1.225)明显高于对照组(0.486±0.442)(P < 0.01);保守性分析显示miR-182-5p成熟体序列在不同物种间高度保守;在线数据库预测获得miR-182-5p潜在的交集靶基因共23个。GO富集结果显示,miR-182-5p靶基因主要参与于锌离子的结合、生物过程的负调控、杂环类化合物的结合、主要代谢过程的调节、GTPase的活性、膜的内在成分的组成、大脑的发育、部分细胞形态的发生、转移酶的活性、信号的调节、泛素结合酶的结合、耳蜗的发育、组蛋白H4-K16的乙酰化作用、咽系统的发育、神经元演化方向的规范、大脑皮层细胞的迁移、大脑皮层细胞的放射状定向迁移等多个生物学过程。KEGG分析结果显示,miR-182-5p靶基因主要参与肾集合管的酸分泌、醚脂类代谢、志贺菌病、细胞的黏附、细菌侵袭上皮细胞等5条信号通路。 结论miR-182-5p在AD病人血清中高表达,其可能通过多条信号通路参与AD的发展过程。 -
关键词:
- 阿尔兹海默病 /
- miR-182-5p /
- 生物信息学 /
- 靶基因
Abstract:ObjectiveTo investigate the expression level of miR-182-5p in the peripheral blood serum of patients with Alzheimer's disease(AD), predict its target genes and conduct functional analysis by bioinformatics methods. MethodsThe dataset of GSE104514 was searched and downloaded from the GEO database. The differentially expressed miRNAs of the dataset were analyzed by GEO2R tool. Among which, miR-182-5p was the most differentially expressed miRNA and was selected for in-depth analysis. Fifteen normal controls(NC) and twenty-eight patients with AD were enrolled in this study. The expression levels of miR-182-5p in peripheral blood serum were determined by real-time fluorescent quantitative PCR. MiRBase database was used to analyze the conservativeness of miR-182-5p among different species. miRcode, miRDB, miRWalk and Target Scan databases were performed to predict the target genes of miR-182-5p, and the common target genes of these databases were screened out. GO and KEGG functional enrichment analysis of miR-182-5p target genes were conducted by DAVID and KOBAS databases. ResultsThe expression of miR-182-5p in brain tissue of AD mice was higher than that in wild mice(P < 0.05). The expression level of miR-182-5p in the peripheral blood serum of AD patients(2.022±1.225) was significantly higher than that of the control group(0.486±0.442)(P < 0.01). Conservative analysis showed that the mature sequence of miR-182-5p was highly conserved in different species. A total of 23 potential intersection target genes of miR-182-5p were predicted by the online databases. The results of GO enrichment analysis suggested that the target genes of miR-182-5p were mainly enriched in zinc ion binding, negative regulation of biological process, heterocyclic compound binding, regulation of primary metabolic process, GTPase activity, intrinsic component of membrane, brain development, cell part morphogenesis, transferase activity, regulation of signaling, ubiquitin conjugating enzyme binding, cochlea development, histone H4-K16 acetylation, pharyngeal system development, neuron fate specification, cerebral cortex cell migration, and cerebral cortex radially oriented cell migration, etc. The results of KEGG signal pathway showed that target genes were mainly enriched in 5 signal pathways, including acid secretion of renal collecting tube, ether lipid metabolism, Shigellosis, adhesion of cell mucosa, and bacterial invasion of epithelial cells. ConclusionsMiR-182-5p is highly expressed in the peripheral blood serum of AD patients and may participate in the development of AD through multiple signaling pathways. -
Key words:
- Alzheimer's disease /
- miR-182-5p /
- bioinformatics /
- target genes
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表 1 miRNAs表达水平的差异
基因名称 B LogFC t P miR-M1-2-3p -3.48 -2.01 3.10 < 0.01 miR-877* -3.74 -2.52 2.69 < 0.05 miR-182 -4.02 2.78 2.25 < 0.05 miR-10a -4.03 -1.20 2.23 < 0.05 miR-10b -4.03 -1.20 2.23 < 0.05 miR-17* -4.09 -1.84 2.13 < 0.05 表 2 不同物种间miR-182-5p的序列比较
序列号 名称 序列 物种 MIMAT0038556 cli-miR-182-5p UUU GGC AAU GGU
AGA ACU CAC ACUColumba livia MIMAT0037544 gga-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACU GGallus gallus MIMAT0036764 oha-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACU GOphiophagus hannah MIMAT0037798 cpi-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUChrysemys picta MIMAT0047699 dno-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUDasypus novemcinctus MIMAT0048285 ocu-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUOryctolagus cuniculus MIMAT0047048 cpo-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUCavia porcellus MIMAT0038194 ami-miR-182-5p UUU GGC AAU GGU
AGA ACU CAC ACUAlligator mississippiensis MIMAT0038980 pbv-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUPython bivittatus MIMAT0000259 hsa-miR-182-5p UUU GGC AAU GGU AGA
ACU CAC ACUHomo sapiens 表 3 miR-182-5p靶基因GO分析结果
GO编号 名称 n P 基因 0008270 zinc ion binding 3 < 0.01 YAF2, ISL1, RBM5 0048519 negative regulation of biological process 4 < 0.01 YAF2, ISL1, RBM5, PAFAH1B1 1901363 heterocyclic compound binding 4 < 0.01 PHF20, ISL1, RBM5, TRIO 0080090 regulation of primary metabolic process 4 < 0.01 ANKIB1, ISL1, PHF20, MEOX1 0051020 gtpase binding 2 < 0.01 TBXA2R, TRIO 0031224 intrinsic component of membrane 4 < 0.01 SLC7A14, TSPAN9, ADAM22, SLC39A9 0007420 brain development 2 < 0.01 ISL1, PAFAH1B1 0032990 cell part morphogenesis 2 < 0.01 ISL1, PAFAH1B1 0023052 signaling 4 < 0.01 TRIO, SYPL1, TSPAN9, PAFAH1B1 0016740 transferase activity 3 < 0.01 PHF20, PDK4, TRIO 0023051 regulation of signaling 3 < 0.01 ISL1, PHF20, PAFAH1B1 0031624 ubiquitin conjugating enzyme binding 1 < 0.01 ANKIB1 0090102 cochlea development 1 < 0.01 PAFAH1B1 0043984 histone h4-k16 acetylation 1 < 0.01 PHF20 0060037 pharyngeal system development 1 < 0.01 ISL1 0048665 neuron fate specification 1 < 0.01 ISL1 0046972 histone acetyltransferase activity(h4-k16 specific) 1 < 0.01 PHF20 0003924 gtpase activity 2 < 0.01 TRIO, PAFAH1B1 0021795 cerebral cortex cell migration 1 < 0.01 PAFAH1B1 0021799 cerebral cortex radially oriented cell migration 1 < 0.01 PAFAH1B1 表 4 miR-182-5p靶基因KEGG分析结果
通路编号 名称 n P 基因编号 hsa04966 Collecting duct acid secretion 1 < 0.05 6521 hsa00565 Ether lipid metabolism 1 < 0.05 5048 hsa05131 Shigellosis 1 < 0.05 7414 hsa04520 Adherens junction 1 < 0.05 7414 hsa05100 Bacterial invasion of epithelial cells 1 < 0.05 7414 -
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