MiR-29b-3p通过GRM4调控前额皮层神经元凋亡研究

Study on the apoptosis of prefrontal cortical neurons regulated by MiR-29b-3p through GRM4

目的研究miR-29b-3p靶向GRM4对前额皮质神经元凋亡的影响及作用机制。方法培养原代前额皮层神经元,转染miR-29b-3p的mimics和inhibitor至原代前额皮层神经元,采用qRT-PCR及WB方法检测miR-29b-3p及GRM4表达水平,MTT法检测神经元细胞活力,流式细胞技术检测神经元细胞凋亡,免疫双标法检测GRM4与凋亡相关蛋白(cleaved caspase-3)关系;采用双荧光素酶法检测miR-29b-3p靶向GRM4基因的机制。结果激活miR-29b-3p表达使前额皮层神经元细胞GRM4表达降低,神经元细胞活力增加,细胞凋亡减少;抑制miR-29b-3p表达使前额皮层神经元细胞GRM4表达增加,神经元细胞活力降低,神经元细胞凋亡增加。免疫双标法结果显示,前额皮层神经元GRM4表达水平与凋亡相关蛋白表达水平呈正相关关系;双荧光素酶法证实GRM4是miR-29b-3p发挥作用的靶点。结论MiR-29b-3p通过抑制GRM4表达降低前额皮层神经元凋亡。

Objective To study the effect and action mechanism of miR-29b-3p on the apoptosis of prefrontal cortical neurons through GRM4.Methods The primary prefrontal cortical neurons were cultured,and then they were transfected with MiR-29b-3p mimics and inhibitor.The expression level of miR-29b-3p and GRM4 were assessed via qRT-PCR and WB methods.The activity of neurons was evaluated with MTT method,the apoptosis of neurons was detected with flow cytometry,the relationship between GRM4 and apoptosis-related protein(cleaved caspase-3)was determined with double immunofluorescence labeling technique;and the mechanism of targeting GRM4 gene by miR-29b-3p was confirmed with double luciferase method.Results The activation of the expression of miR-29b-3p lowered the expression of GRM4 in the prefrontal cortical neurons,increasing the activity of neurons and reducing the apoptosis of neurons.The inhibition of the expression of miR-29b-3p promoted the expression of GRM4,lowering the activity of neurons and promoting the apoptosis of neurons.The double immunofluorescence labeling test showed that the expression of GRM4 in the prefrontal cortical neurons increased with the expression of apoptosis-related protein;double luciferase test confirmed that the GRM4 was the target of miR-29b-3p.Conclusion MiR-29b-3p is able to inhibit the apoptosis of prefrontal cortical neurons by inhibiting the expression of GRM4.