Upregulation of developmentally-downregulated miR-1247-5p promotes neuroprotection and axon regeneration in vivo [RNA-seq]

Numerous micro-RNAs (miRNAs) affect neurodevelopment and neuroprotection, but potential roles of many miRNAs in regulating these processes are still unknown. Here, we used the retinal ganglion cell (RGC) central nervous system (CNS) projection neuron and optic nerve crush (ONC) injury model, to optimize a mature miRNA arm-specific quantification method for characterizing the developmental regulation of miR-1247-5p in RGCs, investigated whether injury affects its expression, and tested whether upregulating miR-1247-5p-mimic in RGCs promotes neuroprotection and axon regeneration. We found that, miR-1247-5p is developmentally-downregulated in RGCs, and is also further downregulated after ONC. Importantly, RGC-specific upregulation of miR-1247-5p promoted neuroprotection and axon regeneration after injury in vivo. To gain insight into the underlying mechanisms, we analyzed by bulk-mRNA-seq embryonic and adult RGCs, along with adult RGCs transduced by miR-1247-5p-expressing viral vector, and identified developmentally-regulated cilial and mitochondrial biological processes, which were reinstated to their embryonic levels in adult RGCs by upregulation of miR-1247-5p. Because axon growth is also a developmentally-regulated process, in which mitochondrial dynamics play important roles, it is possible that miR-1247-5p promoted neuroprotection and axon regeneration through regulating mitochondrial functions. Adult RGCs transduced with AAV2 expressing miR-1247-5p mimic or scramble shRNA mimic were Thy1-immunopanned from single cell retinal suspension (from 10 retinas per condition) and FACS’ed for mCherry+ cells. Approximately, 5,000 RGCs (per condition) were collected by FACS. Approximately, 50,000 of embryonic day 18 (E18) RGCs were Thy1-immunopanned from single cell retinal suspension. RNA was isolated immediately using the Zymo QuickRNA microprep kit. Total RNA with RNA Integrity Number (RIN) ≥ 9 (by Bioanalyzer 2100 using the Nano 6000 kit, Agilent) was extracted using Direct-zol RNA MiniPrep kit (R2050, Zymo Research). cDNA libraries were prepared using polyA-selected RNA (TruSeq RNA Library Prep Kit, Illumina), paired reads sequenced 100 bp from each end on HiSeq 2000 Sequencer (Illumina), passed QC filters, mapped to the mm39 genome and transcriptome by Hisat2, gene expression was normalized as fragments per kilobase of transcript per million mapped reads (FPKM), and analyzed by Cufflinks/CuffDiff.

大量微小RNA（micro-RNAs, miRNAs）可影响神经发育与神经保护，但众多miRNA在调控上述过程中的潜在作用仍未明确。本研究利用视网膜神经节细胞（retinal ganglion cell, RGC）中枢神经系统（central nervous system, CNS）投射神经元及视神经钳夹伤（optic nerve crush, ONC）损伤模型，优化了成熟miRNA臂特异性定量方法，以表征miR-1247-5p在RGC中的发育调控模式；同时探究了损伤是否会影响其表达，并验证在RGC中过表达miR-1247-5p模拟物是否可促进神经保护与轴突再生。
研究发现，miR-1247-5p在RGC中呈发育性下调，且在ONC损伤后进一步下调。值得注意的是，在体损伤后，RGC特异性过表达miR-1247-5p可促进神经保护与轴突再生。
为深入探究潜在分子机制，本研究对胚胎期及成年RGC、经表达miR-1247-5p的病毒载体转导的成年RGC进行了批量mRNA测序（bulk-mRNA-seq），鉴定出发育调控的纤毛与线粒体生物学过程；且通过过表达miR-1247-5p，成年RGC中的上述过程可恢复至胚胎期水平。由于轴突生长同样属于发育调控过程，且线粒体动态在此过程中发挥重要作用，因此miR-1247-5p或可通过调控线粒体功能促进神经保护与轴突再生。
本研究从单个细胞视网膜悬液（每组取材10个视网膜）中通过Thy1免疫淘选获取过表达miR-1247-5p模拟物或乱序shRNA模拟物的腺相关病毒2型（AAV2）转导的成年RGC，并通过荧光激活细胞分选（FACS）分选出mCherry阳性细胞，每组约收集5000个RGC。同时从单个细胞视网膜悬液中免疫淘选得到约50000个胚胎第18天（embryonic day 18, E18）的RGC。
即刻使用Zymo QuickRNA微量制备试剂盒分离RNA。采用Direct-zol RNA微量制备试剂盒（货号R2050, Zymo Research）提取RNA完整性数（RNA Integrity Number, RIN）≥9的总RNA（通过安捷伦Nano 6000试剂盒在2100生物分析仪上检测）。使用polyA富集RNA（TruSeq RNA文库制备试剂盒, Illumina）构建cDNA文库，在HiSeq 2000测序仪（Illumina）上进行双端测序，每端读长100 bp。测序数据通过质控过滤后，利用Hisat2比对至mm39基因组与转录组；基因表达以每百万映射读段每千碱基转录本片段数（FPKM）进行标准化，并通过Cufflinks/CuffDiff进行分析。