Mechanisms Of MiR-466d-3p Through Targeting RIPK3 In Neuronal Cell-Injured Mice With Intracerebral Hemorrhage

Objective: Intracerebral hemorrhage(ICH)is associated with high morbidity and fatality rates.A huge body of evidence suggests that miRNAs play a role in its pathogenesis.We found that miR-466d-3p was differentially under-expressed after brain haemorrhage by sequencing brain haemorrhage and normal group samples using miRNA microarray counting.Indeed,this study clarified the role of miR-466 in ICHrelated neuronal necroptosis and uncovered novel therapeutic targets for this condition.Methods: A brain hemorrhage model was established by injecting collagenase IV into the right basal ganglia of C57BL/6 mice.Intraventricular miR-466 agonist was subsequently administered to the mice.Experiments in cell used a model of cerebral bleeding caused by adding hemin to a line of hippocampus neurons cells(HT-22).Brain edema and neurobehavior were evaluated.The q RT-PCR assay validated that miR466 expression in the intracerebral hemorrhage model.Moreover,the protein levels of RIPK3 and phospho-mixed lineage kinase domain-like protein(p-MLKL)were measured in brain tissue and cells using Western blotting in order to examine alterations following modeling.We assessed cerebral edema and neurological deficits 24 h after the cerebral haemorrhage model.The mice’s spatial memory learning ability was assessed by a water maze experiment starting on day 25 after the mouse brain haemorrhage model.Furthermore,the expression and location of miR466 and RIPK3 were assessed using RNA fluorescence in situ hybridization(FISH)and immunofluorescence double-staining.Lastly,the interaction between miR-466 and RIPK3 was investigated using a dual luciferase reporter gene assay.Results: Analysis of the results by rt-q PCR revealed that the level of miR-466 was downregulated in brain tissue of ICH mice and after HT22 isolated brain hemorrhage model compared with the sham group.After ICH,RIPK3 and p-MLKL levels rose compared to the sham group.miR-466 overexpression down-regulated the expression level of RIPK3 and p-MLKL,limited brain swelling and neurobehavioral abnormalities,and enhanced cognitive functions such as memory and learning.FISH and immunofluorescence double staining revealed the presence of miR-466 and RIPK3 colocalization in the brain tissue around the hematoma of mice after ICH.Dual luciferase reporter gene assay verified the target relationship between miR-466 and the 3’UTR of RIPK3.Conclusion: miR-466 regulates necroptosis by targeting RIPK3 in mice with intracerebral hemorrhage.