MATR3促进m6A甲基化修饰的环状RNA BRIP1形成与核浆重分布在慢性脑缺血神经元铁死亡中的机制

The mechanism of MATR3 promoting m6A methylation-modified circular RNA BRIP1 formation and nucleocytoplasmic redistribution in neuronal ferroptosis in chronic cerebral ischemia

目的研究RNA结合蛋白MATR3及环状RNA(circRNA)BRIP1在慢性脑缺血(CCI)神经元的表达以及OGD诱导的人神经元细胞铁死亡的影响及其相关机制。方法应用Western blot方法检测MATR3在正常神经元和OGD诱导的HNs中的表达;应用Real-time PCR方法检测circBRIP1在正常神经元和OGD诱导的HNs中的表达;应用RNA免疫沉淀实验验证MATR3与circBRIP1的结合作用;应用MeRIP实验验证METTL3与circBRIP1的结合作用;应用CCK8法、脂质过氧化物试剂盒测定方法、C11-BODIPY染色、流式细胞仪方法、谷胱甘肽试剂盒测定方法以及铁测定试剂盒测定方法检测沉默MATR3及circBRIP1对OGD诱导的神经元铁死亡的影响;应用RNA IP实验检测circBRIP1与FSP1的结合作用;应用Western blot方法检测沉默circBRIP1对FSP1蛋白表达水平的影响。结果MATR3和circBRIP3在OGD诱导的HNs中高表达,外源性敲减其表达量能抑制神经元的铁死亡。MATR3能够结合并促进circBRIP3的形成。高表达的METTL3促进circBRIP3m6A甲基化修饰进而促进其核浆重分布。结论MATR3促进circBRIP3形成,m6A甲基化修饰增多的circBRIP3出核调控FSP1蛋白的表达进而调控OGD诱导的HNs铁死亡。

Objective To study the expression of RNA-binding protein MATR3 and circular RNA(circRNA)BRIP1 in neurons of chronic cerebral ischemia(CCI)and CCI-induced human neurons(HNs)effects of ferroptosis and related mechanisms.Methods Western blot was used to detect the expression of MATR3 in normal neurons and CCI-induced HNs.Real-time PCR method was used to detect the expression of circBRIP1 in normal neurons and CCI-induced HNs.RNA immunoprecipitation assay was used to verify the combination of MATR3 and circBRIP1.MeRIP experiment was used to verify the binding effect of METTL3 and circBRIP1.CCK8,lipid peroxide kit,C11-BODIPY staining,flow cytometry,glutathione kit and iron kit were used to determine the influence of MATR3 and circBRIP1 on OGD-induced iron death of neurons.RNA IP assay was used to detect the binding effect of circBRIP1 and FSP1.Western blot was used to detect the effect of silencing circBRIP1 on the expression of FSP1.Results MATR3 and circBRIP3 were highly expressed in CCI-induced HNs,and exogenous knockdown of their expression levels could inhibit neuronal ferroptosis.MATR3 can bind and promote the formation of circBRIP3.The highly expressed METTL3 promotes the methylation of circBRIP3 m6A and promotes its nucleoplasm redistribution.Conclusion MATR3 promotes the formation of circBRIP3,and the nuclear export of circBRIP3 with increased m6A methylation regulates the expression of FSP1 and then regulates CCI-induced ferroptosis in HNs.