MicroRNA-138-5p targets pro-apoptotic factors and favours neural cell survival

Abstract

Background The central nervous system-enriched microRNA miR-138-5p becomes significantly downregulated after spinal cord injury (SCI). miR-138-5p modulates essential biological processes in the Central Nervous System (CNS). It also overcomes apoptosis by inhibiting the expression of proteins, including the effector CASP3, key in different cell death pathways. Therefore, we hypothesize that miR-138-5p downregulation following SCI underlies the overexpression of apoptotic genes and sensitizes neural cells to noxious stimuli. To confirm this hypothesis, this study aims a) to identify and validate miR-138-5p targets among the pro-apoptotic genes overexpressed following SCI; and b) to confirm that the miR-138-5p is able to modulate cell death in neural cells Methods We employed computational tools to identify potential pro-apoptotic targets of miR-138-5p. Dysregulation of selected targets after SCI and its relationship to changes in miR-138-5p expression were analysed through qRT-PCR in a rat SCI model. Validation of the regulation of those apoptotic targets was carried out by luciferase reporter, qRT-PCR, and immunoblot assays in cultures of neural cell lines transfected with a mimetic of the microRNA. The functional effects of modifying the expression of miR-138-5p were later examined in cultures of the rat neural cell line C6 employing enzymatic assays to measure the activity of effector CASP3 and CASP7 together with MTT and flow cytometry assays to estimate cell death. Results Consensus among different algorithms identified 209 potential targets of miR-138-5p. A total of 176 of them become dysregulated after SCI, including proteins basic to apoptosis process such as CASP3 and CASP7, or BAK (Bcl-2 homologous antagonist/killer). Downregulation of miR-138-5p after SCI correlates with the overexpression of these three targets. Cell culture analyses confirm that miR-138-5p targets their 3’UTRs and reduces their expression after microRNA transfection. Transfection of miR-138-5p in C6 cell line results in a reduced effector caspase activity and protects cells from apoptotic stimulation. Conclusions Our results demonstrate that downregulation of miR-138-5p after SCI can be deleterious to spinal neural cells. A mixture of direct effects mediated by the upregulation of apoptotic targets and indirect effects related to the upregulation of cell cycle proteins can be expected.