Upregulation of developmentally-downregulated miR-1247-5p promotes neuroprotection and axon regeneration in vivo [Small-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: Approximately 200,000 of Thy1-immunopanned RGCs were collected (from each condition) and RNA was isolated immediately for preparing cDNA libraries using adaptors that ligate selectively to the endogenously modified 5' and 3' ends of processed small RNAs, thereby enriching specifically for mature small RNAs, including miRNAs, and excluding degraded RNA fragments, precursors, and other RNA species (TruSeq Small RNA Kit, Illumina). Small RNA cDNA library products in the range of 140-160 base pairs (including 5' and 3' adapters and an insert corresponding to a small RNA) were sequenced as paired reads 100 bp from each end on HiSeq 2000 Sequencer (Illumina), passed QC filters, mapped to the mm39 mouse genome and transcriptome by Bowtie, and quantified/analyzed using featureCounts from the subread package and DESeq2.