目的:探讨Rab10基因在抑郁症中的神经保护作用及其介导机制。
方法:48只雄性SD大鼠随机分为对照组和3个慢性不可预知轻度应激(CUMS)组(n=12)。后3组大鼠注射生理盐水,腺相关病毒(AAV)载体,或CUMS建模后侧脑室中Rab10过表达的AAV载体。使用行为测试评估大鼠的抑郁行为变化。TargetScan数据库用于预测与Rab10和结合位点相互作用的miRNA。使用双荧光素酶和放射免疫沉淀(RIP)测定研究了miRNA-103-3p与Rab10之间的相互作用。用CCK-8测定评估皮质酮处理对PC12细胞活力的影响。在皮质酮刺激的PC12细胞中,BDNF的变化,CREB,p62,Beclin-1,Wnt3a,Gsk3β,磷酸化(p)-Gsk3β,用Rab10过表达的AAV载体和miRNA-103-3p抑制剂转染后的β-catenin蛋白表达,单独或组合,使用qRT-PCR和Western印迹进行分析。
结果:侧脑室注射过表达Rab10的AVV载体可明显改善CUMS大鼠的抑郁行为。Rab10的mRNA和蛋白在CUMS大鼠的海马和皮质类固醇表达的PC12细胞中显著下调。生物信息学剖析和双荧光素酶和RIP试验成果证实了miRNA-103-3p与Rab10的靶向关系。在PC12单元格中,过表达Rab10或沉默miRNA-103-3p激活了Wnt/β-catenin信号通路,上调BDNF的表达,CREB和Beclin-1,并下调p62蛋白的表达;沉默Rab10明显阻断miRNA-103-3p抑制剂的作用。
结论:在抑郁症小鼠模型中,miRNA-103-3p通过靶向rab10激活Wnt/β-catenin信号,以改善神经可塑性并促进神经细胞自噬。
OBJECTIVE: To explore the neuroprotective role of Rab10 gene in depression and the mechanism mediating its effect.
METHODS: Forty-eight male SD rats were randomized into a control group and 3 chronic unpredictable mild stress (CUMS) groups (n=12). The rats in the latter 3 groups were subjected to injections of normal saline, an adeno-associated viral (AAV) vector, or a Rab10-overexpressing AAV vector in the lateral ventricle after CUMS modeling. The depressive behavioral changes of the rats were assessed using behavioral tests. The TargetScan database was used to predict the miRNA interacting with Rab10 and the binding sites. The interaction between miRNA-103-3p and Rab10 was investigated using dual-luciferase and radioimmunoprecipitation (RIP) assay. The effect of corticosterone treatment on PC12 cell viability was assessed with CCK-8 assay. In corticosterone-stimulated PC12 cells, the changes in BDNF, CREB, p62, Beclin-1, Wnt3a, Gsk3β, phosphorylated (p)-Gsk3β, and β-catenin protein expressions following transfection with the Rab10-overexpressing AAV vector and a miRNA-103-3p inhibitor, alone or in combination, were analyzed using qRT-PCR and Western blotting.
RESULTS: Injection of Rab10-overexpressing AVV vector into the lateral ventricle significantly improved depressive behaviors of CUMS rats. The mRNA and proteins expression of Rab10 were significantly down-regulated in the hippocampus of CUMS rats and in corticosteronestimulated PC12 cells. Bioinformatics analysis and the results of double luciferase and RIP experiments confirmed the targeting relationship between miRNA-103-3p and Rab10. In PC12 cells, overexpression of Rab10 or silencing miRNA-103-3p activated the Wnt/β-catenin signaling pathway, up-regulated the expressions of BDNF, CREB and Beclin-1, and down-regulated the expression of p62 protein; silencing Rab10 obviously blocked the effect of miRNA-103-3p inhibitor.
CONCLUSIONS: In mouse models of depression, miRNA-103-3p activates Wnt/β-catenin signaling via targeting rab10 to improve neural plasticity and promotes neural cell autophagy.