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Altered miRNA expression of cultured dorsal root ganglia neurons at different times of rat

ABSTRACT: To explored the function of miRNAs during the axon regeneration of rat dorsal root ganglia neurons, we utilized miRCURYTM LNA Array (v.18.0) to find out the altered expression of miRNAs in cultured DRG neurons at different times. When we digest the cells from rat DRGs with enzymes, we only get the cell bodies without axons. During the axons re-growth after the neurons planted, it must be amount of gene and miRNAs will changed. However, we still not fully understand which miRNAs and mRNA will changed and the function of these miRNAs and mRNAs. In addition, this cell model could mimic the regeration of sciatic nerve after injure in vivo, which is still a challenge in clinical.

ORGANISM(S): Mus musculus Rattus norvegicus Homo sapiens

PROVIDER: GSE137837 | GEO | 2021/09/23

REPOSITORIES: GEO

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Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.

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Altered miRNA expression of cultured dorsal root ganglia neurons at different times of rat

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