circ-gabra5通过miR-455-3p调节AKT蛋白加重阿尔茨海默病的认知损害OA

Objective TTo explore the role of circ-gabra5 in the pathogenesis of Alzheimer's disease(AD)in mice.Methods APP/PS1 double transgenic AD mice(6 months old)were selected as the experimental group,and C57BL/6 wild-type(WT)mice were enrolled as the control group.The mice were divided into an AD group and an AD mice with circ-gabra5 knockout(AD+circ-gabra5 knockout)group,with 5 mice in each group.After total RNA was extracted from mouse brain,differential expression of circRNA was detected,and real-time quantitative PCR(RT-qPCR)was used to validate the top 6 circRNA genes with differences.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were performed.Using hippocampal stereo injection of lentivirus to knock out the circ-gabra5 gene,the learning and memory function of mice was detected with Morris water maze test,and the number of Aβ plaques in mouse brain tissue was observed through immunohistochemistry.Transmission electron microscopy was utilized to observe the mitochondrial damage and synaptic count in mouse neurons.Dual luciferase reporter assay and RNA binding protein immunoprecipitation(RIP)assay were employed to verify the interaction between circ-gabra5 and miR-455-3p.Western blotting was apllied to detect the expression level of AKT protein.Results There were 273 differentially expressed genes screened between AD mice and WT mice,including 163 up-regulated and 110 down-regulated genes.Circ-gabra5 had the largest fold difference of 1.39(P<0.05).GO and KEGG analyses suggested that the functional expression differences between AD mice and WT mice were enriched in thyroid hormone,PI3K/AKT,RNA metabolism and other signaling pathways.Knocking out of circ-gabra5 gene in AD mice resulted in obvious improvement in cognitive impairment in Morris water maze test,decreased Aβ plaques by immunohistochemistry,and attenuated neuronal mitochondrial damage and more cortical synapses in AD mice by transmission electron microscopy(P<0.05).According to the starBase database prediction,circ-gabra5 and miR-455-3p have complementary sequences.Dual luciferase reporter assay and RIP assay confirmed the interaction between circ-gabra5 and miR-455-3p.Database bioinformatics analysis predicted that miR-455-3p and AKT have complementary sequences.qPCR indicated that knocking down the circ-gabra5 gene in primary cultured neurons of AD mice resulted in a decrease in miR-455-3p expression,and subsequent Western blot analysis showed a decrease in AKT protein level(P<0.01).Conclusion Circ-gabra5 exacerbates cognitive impairment in AD mice by regulating AKT protein through miR-455-3p,increases Aβ deposition in brain tissue,aggravates neuronal mitochondrial damage,and reduces the number of neural synapses.