胰岛淀粉样多肽对阿尔茨海默病小鼠脑组织中LncRNA和mRNA表达谱的影响

Analysis of long non-coding RNA and mRNA expression profiles in brain tissue of Alzheimer's disease mice after islet amyloid polypeptide intervention

目的探讨胰岛淀粉样多肽(IAPP)对阿尔茨海默病(AD)小鼠脑组织中长链非编码RNA(LncRNA)和信使RNA(mRNA)表达谱的影响。方法选取7月龄雄性APP/PS1转基因AD模型小鼠10只,体质量20~30 g。将AD模型小鼠按数字表法随机分为IAPP干预组和对照组,每组5只。IAPP干预组小鼠腹腔内注射0.5μmol/L的IAPP(200μg/kg),每日1次。对照组小鼠腹腔注射相同剂量的磷酸盐缓冲液。两组小鼠干预时间均为10周。干预完成后,颈椎脱位处死小鼠,开颅剥离完整脑组织提取总RNA。应用基因芯片技术获得2组小鼠脑组织LncRNA和mRNA表达谱,筛选出差异表达的LncRNA和mRNA。在差异表达的LncRNA和mRNA中随机选取6个LncRNA,采用实时定量聚合酶链反应(qRT-PCR)验证基因芯片结果的可靠性。将筛选出的差异表达的mRNA与基因本体论(GO)数据库的各条目比对,从分子功能、生物过程和细胞成分3个领域行GO富集分析;使用京都基因和基因组百科全书(KEGG)数据库行信号通路富集分析。结果实验过程中小鼠死亡4只,每组2只。IAPP干预组与对照组比较,显著差异表达的LncRNA共有902个,其中显著上调238个、显著下调664个;显著差异表达的mRNA共555个,其中显著上调236个、显著下调319个。qRT-PCR检测验证LncRNA的表达情况,与对照组比较,IAPP干预组AD小鼠脑组织中AK034056、Spock3表达上调,Map3k8、rgs20、Rint、Ppp2r1b表达下调,差异均有统计学意义(P值均<0.05)。qRT-PCR检测结果与芯片结果相符。GO富集分析:555个差异表达的mRNA,富集得到2224个GO条目,其中显著上调的差异表达mRNA具有蛋白质结合、细胞周期等分子功能,与催化复合物、蛋白质复合物等细胞组分相关,参与了细胞代谢、高分子代谢等生物过程;显著下调的差异表达mRNA具有G蛋白偶联受体活性、跨膜信号受体活性等相关分子功能,与细胞黏附复合物、整合素复合物等细胞组分相关,参与G蛋白偶联受体信号通路、生物过程的调节等生物学过程。KEGG通路富集分析:555个差异表达的mRNA,富集得到62个通路,其中显著上调的差异表达mRNA富集评分较高的通路为神经退行性变的途径、阿尔茨海默病通路、氧化磷酸化通路、GABA能突触通路等,显著下调的差异表达mRNA富集评分较高的通路为钙信号通路、趋化因子信号通路、磷酸肌醇代谢通路、神经活性配体-受体相互作用通路、白细胞介素-17信号通路等。结论IAPP干预使AD小鼠脑组织LncRNA、mRNA表达谱发生显著变化。差异表达的mRNA分别参与多种不同的生物学过程,差异表达的LncRNA可能通过调控相关mRNA的表达,发挥其生物学功能。

Objective To investigate the changes of long non-coding RNA(LncRNA)and messenger RNA(mRNA)expression profiles in the brain tissue of Alzheimer's disease(AD)mice after islet amyloid polypeptide(IAPP)intervention.Methods Ten 7-month-old male APP/PS1 transgenic AD model mice were selected,weighing 20-30 g.The AD model mice were randomly divided into the IAPP intervention group and the control group according to the number table method,with 5 mice in each group.Mice in the IAPP intervention group were intraperitoneally injected with 0.5μmol/L IAPP,200μg/kg,once a day.The mice in the control group were intraperitoneally injected with the same dose of phosphate buffered saline.The intervention time of the mice in both groups was 10 weeks.After the intervention,the mice were sacrificed by cervical dislocation,and total RNA was extracted from the intact brain tissue by craniotomy.The gene chip technology was used to obtain the LncRNA and mRNA expression profiles of the brain tissues of the two groups of mice,and the differentially expressed LncRNA and mRNA were screened out.Six LncRNAs were randomly selected from the differentially expressed LncRNAs and mRNAs,and real-time quantitative PCR(qRT-PCR)was used to verify the reliability of the gene chip results.The screened differentially expressed mRNAs were compared with the entries in the Gene ontology(GO)database,and the GO enrichment analysis was performed from the three fields of molecular function,biological process and cellular components;using the Kyoto encyclopedia of genes and genomes(KEGG)database Signal pathway enrichment analysis.Results During the experiment,4 mice died,2 mice in each group.Compared with the control group,a total of 902 LncRNAs in the IAPP intervention group had significant differences in expression,238 were significantly up-regulated and 664 were significantly down-regulated;a total of 555 mRNAs were significantly differentially expressed,236 were significantly up-regulated and 319 were significantly down-regulated.qRT-PCR detection verified the expression of LncRNA.Compared with the control group,the expressions of AK034056 and Spock3 in the brain tissue of the AD mice in the IAPP intervention group were up-regulated,and the expressions of Map3k8,rgs20,Rint,and Ppp2r1b were down-regulated,and the differences were statistically significant(all P values<0.05).The qRT-PCR detection results were consistent with the chip results.GO enrichment analysis:555 differentially expressed mRNAs were enriched to obtain 2224 GO entries.The significantly up-regulated differentially expressed mRNAs had molecular functions such as protein binding and cell cycle,were related to cellular components such as catalytic complexes and protein complexes,and participated in biological processes such as cellular metabolism and macromolecular metabolism.The significantly down-regulated differentially expressed mRNA had G protein-coupled receptor activity,transmembrane signaling receptor activity and other related molecular functions,and was related to cell adhesion complexes,integrin complexes and other cellular components;participated in G protein-coupled receptors Signaling pathways,regulation of biological processes and other biological processes.KEGG pathway enrichment analysis:555 differentially expressed mRNAs were enriched for 62 pathways.Significantly up-regulated pathways with higher enrichment scores for differentially expressed mRNAs were neurodegenerative pathways,Alzheimer's disease pathways,oxidative phosphorylation pathways,and GABAergic synaptic pathway,etc.Significantly down-regulated pathways with higher enrichment scores for differentially expressed mRNAs were calcium signaling pathway,chemokine signaling pathway,phosphoinositide metabolism pathway,neuroactive ligand-receptor interaction pathway,interleukin-17 signaling pathway,etc.Conclusion IAPP intervention significantly changes the LncRNA and mRNA expression profiles of AD mice brain tissue.Differentially expressed mRNAs are involved in a variety of biological processes,and differentially expressed lncRNAs may exert their biological functions by regulating the expression of related mRNAs.