慢性铝暴露致认知障碍血清外泌体miRNAs差异表达及作用研究

Differential Expression and Role of Serum Exosomes miRNAs in Cognitive Impairment Caused by Chronic Aluminum Exposure

摘要: 目的：筛选慢性铝暴露致认知障碍人群，生信分析其血清外泌体miRNAs的差异表达，构建铝致认知障碍miRNA-mRNA调控网络图，预测其靶基因。方法：筛选广西铝矿区周边某村庄符合慢性铝暴露常驻60岁以上人群，根据MMSE评分表和血铝水平分为铝致认知障碍组6例和健康对照组6例；提取并鉴定血清外泌体，进行第二代高通量测序，筛选出差异表达的miRNAs；分别运用TargetScan数据库、miWalk数据库、miRDB数据库预测靶基因并取交集，运用Cytoscape软件构建miRNAs-mRNA调控网络图；对同时受到两个以上miRNAs调控的靶基因进行GO和KEGG功能富集分析。结果：成功提取并鉴定血清外泌体；完成第二代高通量测序，按照|logFC| ≥ 1，P值 < 0.05的条件，共筛选出130个差异表达的miRNAs，其中表达下调118个，上调12个；根据文献检索筛选其中的5个miRNAs为目的miRNA，即：hsa-miR-381-3p、hsa-miR-370-3p、hsa-miR-135b-5p、hsa-miR-708-3p、hsa-miR-1289；生信分析构建miRNA-mRNA网络调控图中，共有1170个节点、1232条边，显示共有65个靶基因同时受到两个以上miRNAs的调控；对这65个靶基因进行GO和KEGG功能富集后，显示其主要参与调控的生物过程有3个，即：有丝分裂纺锤体形成、蛋白磷酸化及转录负调控、NDA模板形成；细胞成分包括细胞质、轴突起始段、轴突旁节区、神经元投射等9个方面；分子功能包括蛋白质结合、DNA结合、微管结合、蛋白激酶活性、蛋白质丝氨酸/苏氨酸激酶活性等6个方面；KEGG富集到MAPK信号通路。结论：miR-381-3p、miR-370-3p在铝致认知障碍中表达下调，可能通过调控其靶基因FGF7在铝致认知障碍中发挥重要作用，为铝致认知障碍的诊断提供候选靶点。

Abstract: Objectives: To screen people with cognitive impairment caused by chronic aluminum exposure, analyze the differential expression of serum exosomes miRNAs, construct the miRNA-mRNA regulatory network diagram of cognitive impairment caused by aluminum, and predict its tar-get genes. Methods: People over 60 years old in a village around Guangxi aluminum mining area were selected and divided into 6 aluminum-induced cognitive impairment groups and 6 healthy control groups according to MMSE scale and blood aluminum level. The serum exosomes were extracted and identified, and the second generation high-throughput sequencing was per-formed to screen out the differentially expressed miRNAs. The target genes were predicted by TargetScan database, miWalk database and miRDB database respectively, and the intersection was obtained. The miRNAs-mRNA regulatory network diagram was constructed by Cytoscape software. GO and KEGG function enrichment analysis of target genes was regulated by more than two miRNAs at the same time. Results: Serum exosomes were successfully extracted and identified. The second generation of high-throughput sequencing was completed. According to the condition of |logFC| ≥ 1, P value < 0.05, 130 differentially expressed miRNAs were screened out, of which 118 were down-regulated and 12 were up-regulated. According to the literature search, five miRNAs were selected as the target miRNAs, namely: hsa-miR-381-3p, hsa-miR-370-3p, hsa-miR-135b-5p, hsa-miR-708-3p, HSA-mir-1289. There are 1170 nodes and 1232 edges in the miRNA-mRNA network regulation map constructed by the bio-analysis, which shows that 65 target genes are regulated by more than two miRNAs at the same time. After the enrichment of GO and KEGG functions of these 65 target genes, it shows that there are three bi-ological processes that are mainly involved in regulation, namely, mitotic spindle formation, negative regulation of protein phosphorylation and transcription, and NDA template formation; Cell components include cytoplasm, axon initiation segment, axon paraganglion region and neuron projection. Molecular functions include protein binding, DNA binding, microtubule binding, protein kinase activity and protein serine/threonine kinase activity. KEGG is enriched to MAPK signal pathway. Conclusions: The expression of miR-381-3p and miR-370-3p is down-regulated in aluminum-induced cognitive impairment, which may play an important role in aluminum-induced cognitive impairment by regulating its target gene FGF7, thus providing candidate targets for the diagnosis of aluminum-induced cognitive impairment.