Comprehensive analysis of hippocampal miRNAomes in humans and mice.

MicroRNAs (miRNAs) are small endogenous regulatory RNAs involved in hippocampal functions and lesions. Mouse has been regarded as an effective tool to explore the roles of miRNAs in hippocampus. Although the anatomical structure and physiological function across human and mouse hippocampi are highly conserved, the similarity and difference of hippocampal miRNAomes between the two species have not been well characterized. Our present study represents the first attempt to perform a systematic comparison of the miRNAomes between healthy human and mouse hippocampi using high-throughput sequencing followed by bioinformatic analyses. Contrasting between human and mouse hippocampal miRNAs shows a global similarity of expression pattern; however, the interspecific expression conservation of hippocampal miRNAs is positively associated with the interspecific sequence conservation. Although the type of the abundantly expressed hippocampal miRNAs is almost the same across the two species, only the enriched miRNAs in human hippocampus exhibit potential neuro-related activities. In addition, we also identified a novel miRNA and a species-specific miRNA in human hippocampus whose putative targets are associated with neuro-related activities. Collectively, our results can provide a framework to explore the ability of mice to model the physiological and pathological processes of human hippocampus and also aid in understanding the basis for higher order abilities of learning and memory at the miRNA level. Total RNA from frozen hippocampi (7 humans and 7 mice) was purified using Trizol (Invitrogen, USA) protocol with no modifications. The concentration, purity and integrity were detected by Qubit 2.0 (Life Technologies, USA), Nanodrop (Thermo Scientific, USA), and Agilent 2100 bioanalyzer (Agilent Technologies, USA), respectively. One ÎŒg RNA was used to generate a small RNA sequencing library based on TruSeq Small RNA Sample Prep Kit version 2 (Illumina, USA) according to the manufacturer’s instructions. In brief, T4 RNA ligase was used to ligate RNA 5’ and 3’ adaptors to 5' and 3' ends of RNA, respectively. Adapter-ligated RNA was reverse-transcribed using a RNA RT Primer and the resulting cDNA was amplified in an 11-cycle PCR that used RP1 (RNA PCR Primer) and indexed RP1 primers. PCR products of 140–160 bp were isolated following electrophoresis through a 6% Novex Tris-borate polyacrylamide gel (Life Technologies, USA). Quality of the generated small RNA sequencing library were confirmed using Agilent 2100 bioanalyzer (Agilent Technologies, USA). High-throughput sequencing was carried out based on an Illumina HiSeq2500 system.微小RNA（MicroRNAs, miRNAs）是一类小型内源性调控RNA，参与海马体的功能调控与病变过程。小鼠被广泛视为探究miRNAs在海马体中作用的有效模型生物。尽管人类与小鼠的海马体解剖结构和生理功能高度保守，但二者海马体miRNA组（miRNAome）的异同尚未得到充分阐明。本研究首次尝试利用高通量测序结合生物信息学分析，对健康人类与小鼠海马体的miRNA组进行系统性比较。对比人类与小鼠的海马体miRNA发现，二者的表达模式整体相似；然而，海马体miRNA的种间表达保守性与种间序列保守性呈正相关。尽管两类物种中高丰度表达的海马体miRNA类型几乎一致，但仅人类海马体中富集的miRNA展现出潜在的神经相关活性。此外，本研究还在人类海马体中鉴定出一种新型miRNA以及一种物种特异性miRNA，其潜在靶基因与神经相关活动存在关联。综上，本研究结果可为探究小鼠模拟人类海马体生理与病理过程的能力提供研究框架，同时也有助于从miRNA层面解析人类高级学习记忆能力的分子基础。本研究从冷冻海马体样本（7例人类样本与7例小鼠样本）中提取总RNA，严格按照Trizol（Invitrogen，美国）的标准操作流程进行纯化，未做任何修改。分别通过Qubit 2.0（Life Technologies，美国）、Nanodrop（Thermo Scientific，美国）以及Agilent 2100生物分析仪（Agilent Technologies，美国）检测RNA的浓度、纯度与完整性。取1 μg总RNA，按照TruSeq Small RNA Sample Prep Kit version 2（Illumina，美国）的制造商说明书构建小RNA测序文库。具体步骤如下：使用T4 RNA连接酶分别将RNA的5'与3'接头连接至RNA的5'和3'末端；将连接了接头的RNA通过RNA RT引物进行反转录，得到的cDNA以RP1（RNA PCR引物）与带索引的RP1引物为引物，经11轮PCR扩增；通过6% Novex Tris-硼酸聚丙烯酰胺凝胶电泳分离得到140–160 bp的PCR产物。使用Agilent 2100生物分析仪（Agilent Technologies，美国）验证构建完成的小RNA测序文库质量。最终基于Illumina HiSeq2500平台完成高通量测序。