Increased hippocampal excitability in miR-324-null mice

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3' UTR luciferase assays revealed that some of these genes are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease. RNA was extracted from the hippocampi of 5 miR-324-null and 5 wild-type mice using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific), following the manufacturer's protocol to isolate total RNA, and was sequenced using an Illumina NextSeq 500.微小RNA（microRNAs, miRNAs）是一类非编码RNA，可通过翻译抑制及信使RNA（messenger RNA, mRNA）分子降解的方式下调靶基因的表达。单条微小RNA可特异性靶向大量基因转录本，因此每条微小RNA均可在多条生物学通路中发挥关键作用。miR-324是一种被预测可靶向数千条RNA转录本的微小RNA，其在大脑中的表达量远高于其他所有组织，这提示它可能在一条或多条神经通路中发挥功能。本研究首次报道了全身性miR-324敲除小鼠的相关数据，研究人员在体外实验中于该小鼠的海马体中观察到兴奋性增强及发作间期放电现象。本研究通过RNA测序技术，在miR-324敲除小鼠中筛选出了可能与海马体兴奋性增强相关的差异表达基因；同时通过3'非翻译区（3' untranslated region, 3' UTR）荧光素酶报告基因实验，证实其中部分基因为miR-324全新的直接靶标。对如miR-324这类可影响神经活动的微小RNA进行功能表征，并明确其调控的通路，将有助于我们更深入地理解正常神经功能的相关分子机制，同时也可为神经疾病的治疗提供全新的药物靶点。本研究按照厂商提供的实验流程，使用mirVana miRNA Isolation Kit（Thermo Fisher Scientific）从5只miR-324敲除小鼠及5只野生型（wild-type, WT）小鼠的海马体中提取总RNA，并通过Illumina NextSeq 500测序平台完成RNA测序。