阿尔茨海默病患者环状RNA差异表达初步分析

doi:10.3969/j.issn.1673-8640.2021.11.005

摘要/Abstract

摘要：

目的分析阿尔茨海默病（AD）患者外周血环状RNA（circRNA）的差异表达谱,并预测其下游微小RNA（miRNA）靶点和亲本基因的生物学功能。方法采用基因芯片技术分析3例AD患者（AD组）和3例健康体检者（正常对照组）外周血中circRNA的表达谱,筛选出2个组差异表达的circRNA,并进行生物信息学预测。选取其中2个差异表达的circRNA,采用实时荧光定量聚合酶链反应（RT-qPCR）在30例AD患者和22名正常对照者中进行独立验证。结果筛选出144个AD患者与正常对照者之间存在差异表达（倍数改变>1.5且P<0.05）的circRNA,表达上调和表达下调各72个。基因本体论（GO）富集分析结果显示,差异表达的circRNA的亲本基因主要涉及生殖过程、丝裂原活化蛋白激酶级联反应、T细胞受体信号通路、Toll样受体信号通路等生物学过程;京都基因和基因组数据库（KEGG）富集分析结果显示,差异表达的circRNA的亲本基因主要参与炎症性肠病、血管内皮生长因子（VEGF）、内吞作用、IgG Fc段γ受体（FcγR）介导的吞噬作用等信号通路。选取表达上调、下调的circRNA各1个（hsa_circ_0018905和hsa_circ_0024825）进行验证。RT-qPCR结果显示,AD组hsa_circ_0018905相对表达量显著高于正常对照组（P<0.05）,hsa_circ_0024825相对表达量显著低于正常对照组（P<0.01）,与基因芯片结果一致。结论 AD患者外周血存在差异表达的circRNA,部分差异表达的circRNA可能通过“miRNA海绵”作用参与AD的发生、发展。

关键词: 环状RNA, 微小RNA, 阿尔茨海默病

Abstract:

Objective To investigate expression profiles of circular RNA（circRNA） in peripheral blood of patients with Alzheimer's disease（AD）,and to predict its downstream microRNA（miRNA） targets and the biological function of host gene. Methods Expression profiles of circRNA in peripheral blood from 3 AD patients（AD group） and 3 healthy subjects（healthy control group） were analyzed by microarray assay. Differentially expressed circRNA between the 2 groups were identified,and bioinformatics prediction was performed. Two differentially expressed circRNA were further confirmed by real-time fluorescence quantitative polymerase chain reaction（RT-qPCR） in a validation cohort of 30 AD patients and 22 healthy subjects. Results A total of 144 circRNA（fold change>1.5,P<0.05） were identified to be differentially expressed in AD patients as compared to healthy subjects,which included 72 circRNA upregulated and 72 circRNA downregulated. Gene Ontology（GO）was performed to investigate biological functions of their host genes. Host genes may participate in various biological processes,such as reproductive process,regulation of mitogen-activated protein kinase cascade,regulation of T cell receptor signaling pathway and Toll-like receptor signaling pathway. Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway analysis showed that some vital pathways could also be influenced by the differentially expressed circRNA,including inflammatory bowel disease,vascular endothelial growth factor（VEGF）,endocytosis,gamma receptor for the Fc region of immunoglobulin G（FcγR） mediated phagocytosis. Two differentially expressed circRNA were chosen for further validation,including one upregulated circRNA（hsa_circ_0018905） and one downregulated circRNA（hsa_circ_0024825）. The RT-qPCR results showed that compared to healthy control group the relative expression of hsa_circ_0018905 was upregulated in AD group（P<0.05）,and hsa_circ_0024825 was downregulated between AD group and healthy control group（P<0.01）,which were consistent with the microarray assay results. Conclusions This study identifys a few differentially expressed circRNA between AD patients and healthy subjects,which may exhibit a regulatory role in the occurrence and development of AD by the function as "miRNA sponge".

Key words: Circular RNA, MicroRNA, Alzheimer's disease

中图分类号:

R393

娄小燕, 王涛, 高琼, 路伟, 李丹, 李振华, 林萍. 阿尔茨海默病患者环状RNA差异表达初步分析[J]. 检验医学, 2021, 36(11): 1114-1120.

LOU Xiaoyan, WANG Tao, GAO Qiong, LU Wei, LI Dan, LI Zhenhua, LIN Ping. Preliminary analysis of differentially expressed circRNA of patients with Alzheimer's disease[J]. Laboratory Medicine, 2021, 36(11): 1114-1120.

图/表 5

参考文献 15

[1]	MACLIN J M A, WANG T, XIAO S. Biomarkers for the diagnosis of Alzheimer's disease,dementia Lewy body,frontotemporal dementia and vascular dementia[J]. Gen Psychiatr, 2019, 32(1):e100054.
[2]	KRISTENSEN L S, ANDERSEN M S, STAGSTED L V W, et al. The biogenesis,biology and characterization of circular RNAs[J]. Nat Rev Genet, 2019, 20(11):675-691. DOI URL
[3]	BELOUSOVA E A, FILIPENKO M L, KUSHLINSKII N E. Circular RNA:new regulatory molecules[J]. Bull Exp Biol Med, 2018, 164(6):803-815. DOI URL
[4]	American Psychiatric Association. The diagnostic and statistical manual of mental disorders[M]. 5th ed. Arlington VA: American Psychiatric Publishing, 2014.
[5]	VAN ROSSUM D, VERHEIJEN B M, PASTERKAMP R J. Circular RNAs:novel regulators of neuronal development[J]. Front Mol Neurosci, 2016, 9:74.
[6]	LI Z, LIU S, LI X, et al. Circular RNA in schizophrenia and depression[J]. Front Psychiatry, 2020, 11:392.
[7]	ZHANG S, ZHU D, LI H, et al. Characterization of circRNA-associated-ceRNA networks in a senescence-accelerated mouse prone 8 brain[J]. Mol Ther, 2017, 25(9):2053-2061. DOI URL
[8]	DUBE U, DEL-AGUILA J L, LI Z, et al. An atlas of cortical circular RNA expression in Alzheimer disease brains demonstrates clinical and pathological associations[J]. Nat Neurosci, 2019, 22(11):1903-1912. DOI URL
[9]	LI X, YANG L, CHEN L L. The biogenesis,functions,and challenges of circular RNAs[J]. Mol Cell, 2018, 71(3):428-442. DOI URL
[10]	PIWECKA M, GLAŽAR P, HERNANDEZ-MIRANDA L R, et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function[J]. Science, 2017, 357(6357):eaam8526.
[11]	ZHAO Y, ALEXANDROV P N, JABER V, et al. Deficiency in the ubiquitin conjugating enzyme UBE2A in Alzheimer's disease(AD) is linked to deficits in a natural circular miRNA-7 sponge(circRNA;ciRS-7)[J]. Genes(Basel), 2016, 7(12):116.
[12]	胡国艳, 杨欣, 徐鹏, 等. 早老素1基因circRNA在阿尔茨海默病中的表达[J]. 中国实验诊断学, 2018, 22(10):1821-1825.
[13]	TAN L, YU J T, LIU Q Y, et al. Circulating miR-125b as a biomarker of Alzheimer's disease[J]. J Neurol Sci, 2014, 336(1-2):52-56. DOI URL
[14]	DONG H, LI J, HUANG L, et al. Serum microRNA profiles serve as novel biomarkers for the diagnosis of Alzheimer's disease[J]. Dis Markers, 2015, 2015:625659.
[15]	WU Q, YE X, XIONG Y, et al. The protective role of microRNA-200c in Alzheimer's disease pathologies is induced by beta amyloid-triggered endoplasmic reticulum stress[J]. Front Mol Neurosci, 2016, 9:140.

circRNA名称	差异倍数	P值	亲本基因	染色体定位	表达变化
hsa_circ_0018905	63.47	0.002	SAMD8	chr10	上调
hsa_circ_0007352	40.86	0.005	AKAP17A	chrX	上调
hsa_circ_0006148	7.27	0.048	SAMD8	chr10	上调
hsa_circ_0002292	3.01	0.002	NADK2	chr5	上调
hsa_circ_0002474	2.36	0.042	PDE5A	chr4	上调
hsa_circ_0067209	2.27	0.018	EEFSEC	chr3	上调
hsa_circ_0070805	1.79	0.019	PDE5A	chr4	上调
hsa_circ_0000681	1.67	0.003	PRKCB	chr16	上调
hsa_circ_0052941	1.63	0.009	ATAD2B	chr2	上调
hsa_circ_0071036	1.59	0.021	INPP4B	chr4	上调
hsa_circ_0015980	4.50	0.004	PPP1R12B	chr1	下调
hsa_circ_0024825	4.29	0.005	ST3GAL4	chr11	下调
hsa_circ_0087004	3.63	0.003	CLTA	chr9	下调
hsa_circ_0035197	3.28	0.001	ATP8B4	chr15	下调
hsa_circ_0001707	3.28	0.002	ABCA13	chr7	下调
hsa_circ_0029410	2.72	0.004	GLT1D1	chr12	下调
hsa_circ_0004662	2.45	0.023	SOD2	chr6	下调
hsa_circ_0029405	1.82	0.017	GLT1D1	chr12	下调
hsa_circ_0006435	1.78	0.019	UGCG	chr9	下调
hsa_circ_0038799	1.69	0.010	XPO6	chr16	下调

circRNA名称	差异倍数	P值	亲本基因	染色体定位	表达变化
hsa_circ_0018905	63.47	0.002	SAMD8	chr10	上调
hsa_circ_0007352	40.86	0.005	AKAP17A	chrX	上调
hsa_circ_0006148	7.27	0.048	SAMD8	chr10	上调
hsa_circ_0002292	3.01	0.002	NADK2	chr5	上调
hsa_circ_0002474	2.36	0.042	PDE5A	chr4	上调
hsa_circ_0067209	2.27	0.018	EEFSEC	chr3	上调
hsa_circ_0070805	1.79	0.019	PDE5A	chr4	上调
hsa_circ_0000681	1.67	0.003	PRKCB	chr16	上调
hsa_circ_0052941	1.63	0.009	ATAD2B	chr2	上调
hsa_circ_0071036	1.59	0.021	INPP4B	chr4	上调
hsa_circ_0015980	4.50	0.004	PPP1R12B	chr1	下调
hsa_circ_0024825	4.29	0.005	ST3GAL4	chr11	下调
hsa_circ_0087004	3.63	0.003	CLTA	chr9	下调
hsa_circ_0035197	3.28	0.001	ATP8B4	chr15	下调
hsa_circ_0001707	3.28	0.002	ABCA13	chr7	下调
hsa_circ_0029410	2.72	0.004	GLT1D1	chr12	下调
hsa_circ_0004662	2.45	0.023	SOD2	chr6	下调
hsa_circ_0029405	1.82	0.017	GLT1D1	chr12	下调
hsa_circ_0006435	1.78	0.019	UGCG	chr9	下调
hsa_circ_0038799	1.69	0.010	XPO6	chr16	下调

circRNA名称	MRE1	MRE2	MRE3
上调
hsa_circ_0018905	hsa-miR-505-5p	hsa-miR-16-1-3p	hsa-miR-636
hsa_circ_0007352	hsa-miR-2682-3p	hsa-miR-6836-5p	hsa-miR-6132
hsa_circ_0006148	hsa-miR-141-5p	hsa-miR-98-3p	hsa-let-7f-1-3p
hsa_circ_0002292	hsa-miR-8087	hsa-miR-3972	hsa-miR-335-3p
hsa_circ_0002474	hsa-miR-670-3p	hsa-miR-583	hsa-miR-455-3p
hsa_circ_0067209	hsa-miR-8082	hsa-miR-146b-3p	hsa-miR-6876-3p
hsa_circ_0070805	hsa-miR-29b-1-5p	hsa-miR-670-3p	hsa-miR-29a-5p
hsa_circ_0000681	hsa-miR-181c-5p	hsa-miR-181d-5p	hsa-miR-181a-5p
hsa_circ_0052941	hsa-miR-372-3p	hsa-miR-93-5p	hsa-miR-106a-5p
hsa_circ_0071036	hsa-miR-376a-3p	hsa-miR-489-3p	hsa-miR-29b-3p
下调
hsa_circ_0015980	hsa-miR-149-5p	hsa-miR-8057	hsa-miR-449b-3p
hsa_circ_0024825	hsa-miR-708-5p	hsa-miR-1229-5p	hsa-miR-4640-5p
hsa_circ_0087004	hsa-miR-5193	hsa-miR-877-3p	hsa-miR-6760-5p
hsa_circ_0035197	hsa-miR-605-3p	hsa-miR-101-5p	hsa-miR-31-5p
hsa_circ_0001707	hsa-miR-22-5p	hsa-miR-22-3p	hsa-miR-599
hsa_circ_0029410	hsa-miR-197-3p	hsa-miR-412-3p	hsa-miR-146b-3p
hsa_circ_0004662	hsa-miR-4520-2-3p	hsa-miR-208b-5p	hsa-miR-4713-5p
hsa_circ_0029405	hsa-miR-764	hsa-miR-19a-3p	hsa-miR-9-5p
hsa_circ_0006435	hsa-miR-335-3p	hsa-miR-4464	hsa-miR-942-5p
hsa_circ_0038799	hsa-miR-429	hsa-miR-200b-3p	hsa-miR-200c-3p

circRNA名称	MRE1	MRE2	MRE3
上调
hsa_circ_0018905	hsa-miR-505-5p	hsa-miR-16-1-3p	hsa-miR-636
hsa_circ_0007352	hsa-miR-2682-3p	hsa-miR-6836-5p	hsa-miR-6132
hsa_circ_0006148	hsa-miR-141-5p	hsa-miR-98-3p	hsa-let-7f-1-3p
hsa_circ_0002292	hsa-miR-8087	hsa-miR-3972	hsa-miR-335-3p
hsa_circ_0002474	hsa-miR-670-3p	hsa-miR-583	hsa-miR-455-3p
hsa_circ_0067209	hsa-miR-8082	hsa-miR-146b-3p	hsa-miR-6876-3p
hsa_circ_0070805	hsa-miR-29b-1-5p	hsa-miR-670-3p	hsa-miR-29a-5p
hsa_circ_0000681	hsa-miR-181c-5p	hsa-miR-181d-5p	hsa-miR-181a-5p
hsa_circ_0052941	hsa-miR-372-3p	hsa-miR-93-5p	hsa-miR-106a-5p
hsa_circ_0071036	hsa-miR-376a-3p	hsa-miR-489-3p	hsa-miR-29b-3p
下调
hsa_circ_0015980	hsa-miR-149-5p	hsa-miR-8057	hsa-miR-449b-3p
hsa_circ_0024825	hsa-miR-708-5p	hsa-miR-1229-5p	hsa-miR-4640-5p
hsa_circ_0087004	hsa-miR-5193	hsa-miR-877-3p	hsa-miR-6760-5p
hsa_circ_0035197	hsa-miR-605-3p	hsa-miR-101-5p	hsa-miR-31-5p
hsa_circ_0001707	hsa-miR-22-5p	hsa-miR-22-3p	hsa-miR-599
hsa_circ_0029410	hsa-miR-197-3p	hsa-miR-412-3p	hsa-miR-146b-3p
hsa_circ_0004662	hsa-miR-4520-2-3p	hsa-miR-208b-5p	hsa-miR-4713-5p
hsa_circ_0029405	hsa-miR-764	hsa-miR-19a-3p	hsa-miR-9-5p
hsa_circ_0006435	hsa-miR-335-3p	hsa-miR-4464	hsa-miR-942-5p
hsa_circ_0038799	hsa-miR-429	hsa-miR-200b-3p	hsa-miR-200c-3p