MicroRNA-125b mimic inhibits ischemia reperfusion-induced neuroinflammation and aberrant p53 apoptotic signalling activation through targeting TP53INP1

Abstract

Background: Ischemia reperfusion (IR) injury affects neuronal function through multiple pathogeneses that induce neuroinflammation and cellular apoptosis. The important roles of microRNAs (miRs) in the regulation of spinal cord IR have been recently reported. Among these roles, we investigated whether miR-125b and its downstream targets regulated the p53 signalling network and participated in both inflammation and apoptosis.

Methods: An IR model was established via 12-min occlusion of the aortic arch. The direct interaction between miR-125b and TP53INP1 was demonstrated by Western blotting and luciferase assays. The cellular distributions of TP53INP1 were visualised by double immunofluorescence labelling. The effects of miR-125b on the expression of TP53INP1, p53 and release of proinflammatory cytokines were evaluated by synthetic miRs. Additionally, the detection of hind-limb motor function in vivo and motor neuronal apoptosis in vitro were evaluated to explore the potential mechanisms.

Results: IR-induced alterations in hind-limb motor function were closely related to the temporal changes in miR-125b and TP53INP1 expression. The miR-125b/TP53INP1 gene pair was confirmed by luciferase assay. Compared with Sham controls, IR treatment resulted in increased TP53INP1 immunoreactivity that was primarily distributed in neurons. Treatment with miR-125b mimic markedly decreased the protein levels of TP53INP1, p53 and cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, whereas miR-125b control or inhibitor did not have the above-mentioned effects. Moreover, miR-125b mimic improved motor function in vivo and attenuated neuronal apoptosis in vitro, as demonstrated by the increased average Tarlov scores in lower limbs and lower percentages of neurons in the A4 and A2 quadrants of flow cytometry. Fluorescent staining and quantification further indicated that miR-125b mimic decreased the immunoreactivities of p53 and cleaved caspase 3 in neurons and simultaneously reduced the number of double-labelled cells with TP53INP1.

Conclusions: miR-125b mimic partially protected neurons against neuroinflammation and aberrant p53 network activation-induced apoptosis during IR injury through downregulation of TP53INP1.

Keywords: Apoptosis; Ischemia reperfusion injury; MicroRNAs; P53 signalling network; Tumour protein 53-induced nuclear protein 1.