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. Overall design: 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.